Polypeptides and immunizing compositions containing gram positive polypeptides and methods of use

ABSTRACT

The present invention provides isolated polypeptides isolatable from a  Staphylococcus  spp. Also provided by the present invention are compositions that include one or more of the polypeptides, and methods for making and methods for using the polypeptides.

CONTINUING APPLICATION DATA

This application is a continuation patent application of U.S. patentapplication Ser. No. 13/362,992, filed on Jan. 31, 2012, which is acontinuation patent application of U.S. patent application Ser. No.12/272,021, filed on Nov. 17, 2008 (now U.S. Pat. No. 8,709,760), whichis a continuation of U.S. patent application Ser. No. 11/353,459, filedon Feb. 14, 2006 now U.S. Pat. No. 8,007,811), which claims the benefitof U.S. Provisional Application No. 60/652,843, filed Feb. 14, 2005,each of which is incorporated by reference herein.

SEQUENCE LISTING

This application contains a Sequence Listing electronically submittedvia EFS-Web to the United States Patent and Trademark Office as an ASCIItext file entitled “29300380106SequenceListg_ST25.txt,” having a size of85 kilobytes and created on Apr. 9, 2015. The information contained inthe Sequence Listing is incorporated by reference herein.

BACKGROUND

Gram-positive bacteria are a remarkably diverse group of organisms thatcause a variety of diseases in both humans and animals. Some of thepathogens recognized as important in human and/or animal health includebacteria belonging to the families of Corynebacteriaceae,Enterococcacae, Micrococcaceae, Mycobacteriaceae, Nocardiaceae, andPeptococcaceae, which include such bacterial species as Actinomycesspp., Bifidobacterium spp., Corynebacterium spp., Enterococcus spp.,Erysipelothrix spp., Eubacterium spp., Kytococcus spp., Lactobacillusspp., Micrococcus spp., Mobiluncus spp., Mycobacteria spp.,Peptostreptococcus spp., Propionibacterium spp., and Staphylococcus spp.These pathogens cause a multitude of clinical manifestations in manydifferent animal species. The treatment for such infections hashistorically been antibiotics that attack the common structures andfunctions of gram-positive organisms. However, many of the moreubiquitous gram-positive organisms have developed resistance to severalclasses of antibiotics, making treatment of infections difficult. Thewidespread use of antibiotics in the treatment of bacterial diseases inboth humans and food production animals is likely a major contributingfactor in the proliferation of antibiotic-resistant strains of manyspecies of gram-positive organisms. Therefore, there is a great need tofind different treatments that prevent or eliminate infections bygram-positive organisms in animals as well as humans.

Staphylococcal Infections in Agricultural Animals

In the agricultural industry a number of important diseases are causedby gram-positive organisms. Examples of clinical conditions caused bygram positive bacterial infections include, mastitis, septicemia,pneumonia, osteomyelitis, meningoencephalitis, lymphangitis, dermatitis,genital tract infections, metritis, perinatal disease, pituitaryabscesses, arthritis, bursitis, orchitis, cystitis and pyelonephritis,caseous lymphadenitis, tuberculosis, ulcerative lymphangitis,erysipelas, laminitis, tyzzer's disease, tetanus, botulism, enteritis,malignant edema, braxy, bacillary hemoglobinuria, enterotoxemia.Staphylococcus spp., in particular, are capable of infecting manydifferent species of agricultural animals and can cause enormouseconomic losses. For example, the United States dairy industry isestimated to lose approximately $185 per cow annually due to mastitis, adisease often caused by Staphylococcus aureus. Since there are 9.5million head of milking cows in the U.S., the annual cost of mastitis isapproximately $1.8 billion. This is approximately 10% of the total valueof farm milk sales, and about two-thirds of this loss is due to reducedmilk production in sub-clinically infected cows. Other losses are due todiscarded abnormal milk and milk withheld from cows treated withantibiotic, costs of early replacement of affected cows, reduced salevalue of culled cows, costs of drugs and veterinary services, andincreased labor costs. In addition to its prevalence within the bovinedairy industry, mastitis caused by gram-positive cocci is also commonamong goats and sheep. Additional animal diseases caused by S. aureusinclude botryomycosis in horses, purulent synovitis and osteomyelitis inpoultry, snuffles in rabbits, abortions in swine, and tick pyemia inlambs. Other species of staphylococci are major skin pathogens of canine(S. intermedius) and swine (S. hycius). In poultry species,staphylococcal pathogens cause endorcarditis and septicemia.

Staphylococcal Infections in Humans

Staphylococcus spp. are also human pathogens causing a wide variety ofinfections. Use species Staphylococcus aureus, a common colonizer ofhuman mucosa and skin, is an opportunistic pathogen that can causediverse human infectious. For example, S. aureus is the causative agentof several skin infections, including impetigo, furunculosis,cellulitus, and scalded skin syndrome, as well as potentially fatalpost-surgical wound infections. In addition, the exposure ofimmunocompromised individuals to S. aureus in hospital settings hasresulted in organ infections such as pneumonia, urinary tractinfections, osteomyelitis, arthritis, bacteremia, and endocarditis. S.aureus is also the causative agent of toxinoses, most notably toxicshock syndrome and food poisoning. Food poisoning caused by thestaphylococcal enterotoxin B is the most common cause of food-borneillness, surpassing even salmonellosis, campylobacteriosis andlisteriosis. Other species of staphylococci also cause human disease; S.epidermidis, S. haemolyticus and S. hominis commonly infect implantedmedical devices and S. saprophyticus is associated with urinary tractinfections is women.

Virulence Mechanisms of Staphylococci

Staphylococci infect a variety of host tissues and evade the immunesystem through the production of several types of secreted proteins,surface expressed virulence factors and metabolic systems designed forsurvival amidst the limited resources and active defenses associatedwith the host environment. Colonization is the necessary first step inestablishing infection; numerous factors including capsule, lipoteichoicacid, and teichoicc acid are common structural components contributingto colonization. In addition, surface proteins such as staphylococcalfibronectin-binding protein and bone-sialoprotein binding proteinsspecifically bind host tissue components. Toxins are commmonly producedamong staphylococcal pathogens and are highly damaging; several humandiseases, including food poisoning, toxic shock syndrome and exfoliativeskin conditions, are the direct result of extracellular secreted toxinproteins. A single isolate may encode genes for 20-30 different secretedtoxins. Some of the secreted protein products are superantigens that canbind nonspecifically to the MHC class II molecule of anantigen-presenting cell and, simultaneously, to the T-cell receptor of aT cell. The binding induces T cell signaling and leads to the release ofhigh levels of proinflammatory factors, ultimately inducing host damagedue to the overwhelming immune response. Another class of virulencefactors expressed on the surface disguise the bacteria from the hostimmune system. For example, the S. aureus surface-expressed Protein Ainhibits opsonization and phagocytosis by binding of the Fc component ofhost antibody. Numerous proteases, hemolysins (alpha, beta, gamma anddelta), nucleases, lipases, hyaluronidase, and collagenase also aidbacteria in extracting nutrients from surrounding cells and protectingthem against host defenses.

Antibiotic Resistance Among Staphylococci

The CDC estimates that each year nearly 2 million people in the UnitedStates acquire a nosocomial infection, resulting in 90,000 deathsannually. Of these fatal infections, 70% are caused byantibiotic-resistant bacteria. The increase in antibiotic-resistanceamong microbial species is particularly pronounced in skin and mucosalcolonizers such as S. aureus. For example, the vast majority of S.aureus isolated from hospital settings are resistant to penicillin, and50% are also resistant to the semisynthetic penicillins, such asmethicillin, nafcillin, and oxacillin. These isolates, referred to asMRSA (methicillin resistant S. aureus) were first seen in the 1970s, andare now firmly established in hospital settings. Recently there havebeen several cases of MRSA infections in the community, where theinfected individuals had no previous exposure to hospitals or healthcareworkers. This alarming trend is intensified by the isolation of MRSAisolates that are less susceptible to vancomycin, a glycopeptide used totreat MRSA. Very few strains have been shown to be truly resistant tovancomycin according to the CDC's definition of vancomycin resistance,but several MRSA strains have been characterized as consisting ofsubpopulations with reduced susceptibility to vancomycin, or VISA(vancomycin intermediate S. aureus). Since the isolation of vancomycinresistant and vancomycin intermediate strains is a relatively newdevelopment, there is little data concerning their prevalence inhospitals and/or the community. Occasionally, VRSA (vancomycin resistantS. aureus) with full resistance to vancomycin and carrying a resistanceplasmid likely acquired from Enterococcus spp. have also been recoveredfrom humans.

Strategies for the Prevention and Treatment of Staphylococcus Infections

The emergence of numerous gram-positive pathogens that are resistant tomultiple antibiotics has fueled research efforts aimed at developingpreventative vaccines to protect against disease. Vaccines are designedto be administered to patients in order to elicit a long-term memoryresponse from the immune system, so that if the pathogen is encounteredat a future time, the immune system can more quickly and efficientlyclear the pathogen. To date, a broadly-protective vaccine againstgram-positive pathogens associated with a number of severe humandiseases, particularly those disease associated with staphylococcalinfections, is not available. Vaccine development approaches for theprevention of staphylococcal infections include those reporting the useof microbial surface components recognizing adhesion matrix molecules[MSCRAMMS (Nilsson et al. 1998. J Clin Invest 101:2640-9; Menzies et al.2002. J Infect Dis 185:937-43; Fattom et al. 2004. Vaccine 22:880-7],surface polysaccharides (McKenney et al. 2000; McKenney et al. 1999.Science 284:1523-7; Maira-Litran et al. 2002. Infect Immun 70:4433-40;Maira-Litran et al. 2004. Vaccine 22:872-9; Maira-Litran et al. 2005Infect Immun 73:6752-62) and mutated exoproteins (Lowell et al. 1996.Infect Immun 64:4686-93; Stiles et al. 2001. Infect Immun 69:2031-6;Gampfer et al. 2002. Vaccine 20:3675-84), as antigens in subunit vaccinecompositions, as well as one live avirulent strain (Reinoso et al. 2002.Can J Vet Res 66:285-8) and several DNA vaccine approaches (Ohwada etal. 1999. J Antimocrob Chemother 44:767-74); Brouillette et al. 2002.Vaccine 20:2348-57; Senna et al. 2003. Vaccine 21:2661-6). Although manyof these compositions have shown some degree of protection, they haveachieved little cross-protection against diverse staphylococcal strainsand have additionally failed to elicit substantial immune responses inimmunocompromised patients, an important at-risk population fornosocomial infections.

The most severe staphylococcal diseases are those mediated by theaforementioned supermantigenic pyrogenic exotoxins (SPEs) thatnonspecifically stimulate T-cells independent of antigen presentation.Such diseases include toxic shock syndrome, exfoliative skin disease,and possibly Kawasaki syndrome. For these SPE-mediated diseases,immunotherapeutic agents that boost the immune system during an activeinfection are often more effective than vaccines, which are typicallyadministered prior to injection. The overwhelming nature of the immuneresponse to SPE necessitates rapid reduction in toxin activity as thefirst objective in therapy. To date, toxin neutralisation in S.aureus-mediated disease has been most effectively accomplished by theadministration of intravenous human immunoglobulin (IVIG), a purified,concentrated human antibody preparation from several thousand humandonors (Takei et al. 1993. J Clin Invest 91:602-7; Stohl and Elliot.1996, Clin Immunol Immunopathol 79:122-33). The widespread distributionof S. aureus, which colonizes approximately 30% of healthy human adults,coincides with high exposure rates for the majority of the population,so the level of anti-staphylococcal anti-toxin antibodies in IVIG isoften sufficient to neutralize toxin long enough to stabilize the immuneresponse until the bacterial load is reduced with antibiotics(Schlievert, 2001. J Allergy Clin Immunol 108(4 Suppl):S107-110). IVIGpreparations from multiple manufacturers have been shown to neutralizetoxin in proliferation assays with human peripheral blood mononuclearcells, inhibit toxin-induced human T cell-driven B cell differentiationin vitro (Stohl and Elliot. 1996. Clin Immunol Immunopathol 79:122-33;Stohl and Elliott. 1995. J Immunol 155:1838-50; Stohl et al 1994. JImmunol 353:117-27) and reduce IL-4 and IL-2 secretion in PBMCsstimulated with staphylococcal enterotoxin B (Takei et al 1993. J ClinInvest 91:602-7; Darenberg et al. 2004. Clin Infect Dis 38:836-42). IVIGtherapy, with its proven ability to neutralize SPE, is now a recommendedtherapy for Kawasaki syndrome and is gaining favor as a treatment methodfor staphylococcal toxic shock syndrome (Schlievert 2001. J Allergy ClinImmunol 108(4 Suppl):S107-110). Use of IVIG as an immunoprotective woundlavage during surgery has also been investigated in mice (Poelstra etal. 2000. Tissue Eng 6(4:401-411). Although standard IVIG has utilityfor limiting the advance of some staphylococcal SPE-mediated disease,the safety, efficacy and consistency of human IVIG preparationsgenerated from thousands of unselected human donors remainscontroversial (Baker et al. 1992. N Engl J Med 327:213-9; Miller et al.2001. J Allergy Clin Immunol 108:S91-4; Sacher, 2001. J Allergy ClinImmunol 108:S139-46; Darenberg et al. 2004. Clin Infect Dis 38:836-42).Furthermore, the benefit of IVIG in preventing some staphylococcalinfections is doubtful (Baker et al. 1992. N Engl J Med 327:213-9; Hill,H. R. 2000. J Pediatr 137:595-7; Darenberg et al. 2004. Clin Infect Dis38:836-42). In order to increase the effectiveness of IVIG in treatingstaphylococcal infections in certain at-risk populations, aplasma-derived, donor-selected, polyclonal anti-staphylococcal human IgGwith high titers of antibody directed toward the staphylococcal MSCRAMMSclumping factor A (ClfA) and fibrinogen-binding protein G (SdrG) wascreated and tested with success in very low birthweight infants toprevent stephylococcal sepsis (Vernachio et al. 2003. Antimicrob AgentsChemother 47:3400-6; Bloom et al. 2005. Pediatr Infect Dis J 24:858-866;Capparelli et al. 2005. Antimicrob Agents Chemother 49:4121-7). Aspecific humanized monoclonal antibody toward the S. aureus MSCRAMMClumping factor A, is also being developed. The antibody was selectedfrom a pool of thousands of murine anti-ClfA antibodies for its abilityto bind ClfA in a manner that abrogates S. aureus binding to humanfibronectin and was subsequently humanized by mutating specific targetedresidues to mimic the homologous human germline subgroup antibody (Hallet al. 2003. Infect Immun 71:6864-70; Domanski et al. 2005. Infect Immun73:5229-32). The specific antibody is being designed for use inconjunction with antibiotics for the treatment of severelife-threatening S. aureus infection, although animal studies alsodemonstrated a prophylactic protective effect.

SUMMARY

The present invention provides compositions including two or moreisolated polypeptides. The two isolated polypeptides may have amolecular weight of 88 kDa, 55 kDa, 38 kDa, 37 kDa, 36 kDa, 35 kDa, 33kDa, or a combination thereof. For instance, a composition may includeisolated proteins of 88 kDa and 35 kDa. In some aspects the compositionmay include isolated polypeptides having molecular weights of 88 kDa, 55kDa, 38 kDa, 37 kDa, 36 kDa, 35 kDa, and 33 kDa. The molecular weight isdetermined by electrophoresis on a sodium dodecyl sulfate-polyacrylamidegel. The polypeptides are isolatable from a Staphylococcus aureus whenincubated in media including an iron chelator and not isolatable whengrown in the media without the iron chelator. The compositions protectsan animal, such as a mouse or cow or human, against challenge with an S.aureus strain, for instance ATCC strain 19636. The composition mayfurther include a pharmaceutically acceptable carrier, and may furtherinclude an isolated polypeptide having a molecular weight of 150 kDa,132 kDa 120 kDa, 75 kDa, 58 kDa, 50 kDa, 44 kDa, 43 kDa, 41 kDa, 40 kDa,or a combination thereof, and isolatable from a S. aureus when grown inthe media without the iron chelator. In some aspects the polypeptides ofthe composition may be isolated from S. aureus ATCC strain 19636.

The present invention also provides methods for using the compositions.In one aspect the method is for treating in infection in a subject, andincludes administering an effective amount of a composition of thepresent invention to a subject having or at risk of having an infectioncaused by a Staphylococcus spp. In another aspect, the method is fortreating a symptom in a subject, and it includes administering aneffective amount of a composition of the present invention to a subjecthaving an infection caused by a Staphylococcus spp. The subject may be amammal, such as a human, horse or cow. The Staphylococcus spp, may be S.aureus.

The present invention further provides methods for using antibody, forinstance, polyclonal antibody, that specifically binds polypeptides ofthe present invention. In one aspect, the method is for treating aninfection in a subject, and includes administering an effective amountof a composition to a subject having or at risk of having an infectioncaused by a Staphylococcus spp., wherein the composition includesantibody that specifically binds two isolated polypeptides of thepresent invention. In another aspect, the method is for treating asymptom in a subject, and includes administering an effective amount ofa composition to a subject having an infection caused by aStaphylococcus spp., wherein the composition includes antibody thatspecifically binds two isolated polypeptides of the present invention.The subject may be a mammal, such as a human, horse, or cow. TheStaphylococcus spp. may be S. aureus.

Also provided by tire present invention are methods for decreasingcolonization in a subject. In one aspect, the method includesadministering an effective amount of a composition of the presentinvention to a subject colonized by a Staphylococcus spp. In anotheraspect, the method includes administering an effective amount of acomposition to a subject colonized by Staphylococcus spp., wherein thecomposition includes antibody that specifically binds two isolatedpolypeptides of the present invention.

The present invention provides a kit for detecting antibody thatspecifically binds a polypeptide. The kit includes, in separatecontainers, an isolated polypeptide of the present invention, and areagent that detects an antibody that specifically binds thepolypeptide.

The present invention further provides a composition including twoisolated polypeptides having molecular weights selected from 88 kDa, 55kDa, 38 kDa, 37 kDa, 36 kDa, 35 kDa, and 33 kDa, wherein molecularweight is determined by electrophoresis on a sodium dodecylsulfate-polyacrylamide gel. Each polypeptide of the composition has amass fingerprint of at least 80% similarity to a mass fingerprint of apolypeptide of the same molecular weight polypeptide expressed byStaphylococcus aureus ATCC strain 19636, wherein the polypeptide isisolatable from a Staphyhcoccus aureus when incubated in mediacomprising an iron chelator and not isolatable when grown is the mediawithout the iron chelator. For instance, the isolated polypeptide with amolecular weight of 88 kDa has a mass fingerprint of at least 80%similarity to a mass fingerprint of a 88 kDa polypeptide expressed byStaphylococcus aureus ATCC strain 19636, and the isolated polypeptidewith a molecular weight of 55 kDa has a mass fingerprint of at least 80%similarity to a mass fingerprint of a 55 kDa polypeptide expressed byStaphylococcus aureus ATCC strain 19636.

BRIEF DESCRIPTION OP THE FIGURES

FIG. 1. The electrophoretic profile of the proteins of different strainsStaphylococcus aureus derived from different species grown with andwithout iron (lanes marked Fe++ and DP, respectively).

FIG. 2. The difference in mortality between vaccinated andnon-vaccinated mice after homologous and heterologous challenge withStaphylococcus aureus.

FIG. 3. Kaplan-Meier survival curve showing percent survival aftervaccination and homologous challenge with S. aureus ATCC 19636.

FIG. 4. Kaplan-Meier survival curve showing percent survival aftervaccination and heterologous challenge with S. aureus ATCC 19636.

FIG. 5. The Kaplan-Meier survival curve showing percent survival afterpassive immunization and homologous challenge with S. aureus ATCC 19636.

FIG. 6. The Kaplan-Meier survival curve showing percent survival afterpassive immunisation and heterologous challenge with S. aureus strain1477.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

The present invention provides polypeptides and compositions includingpolypeptides. As used herein, “polypeptide” refers to a polymer of aminoacids linked by peptide bonds. Thus, for example, the terms peptide,oligopeptide, protein, and enzyme are included within the definition ofpolypeptide. This term also includes post-expression modifications ofthe polypeptide, such as glycosylations, acetylations, phosphorylations,and the like. The term polypeptide does not connote a specific length ofa polymer of amino acids. A polypeptide may be isolatable directly froma natural source, or can be prepared with the aid of recombinant,enzymatic, or chemical techniques. In the case of a polypeptide that isnaturally occurring, such a polypeptide is typically isolated. An“isolated” polypeptide is one that has been removed from its naturalenvironment. For instance, an isolated polypeptide is a polypeptide thathas been removed from the cytoplasm or from the membrane of a cell, andmany of the polypeptides, nucleic acids, and other cellular material ofits natural environment are no longer present. An “isolatable”polypeptide is a polypeptide that could be isolated from a particularsource. A “purified” polypeptide is one that is at least 60% free,preferably at least 75% free, and most preferably at least 90% free fromother components with which they are naturally associated. Polypeptidesthat are produced outside the organism in which they naturally occur,e.g., through chemical or recombinant means, are considered to beisolated and purified by definition, since they were never present in anatural environment. As used herein, a “polypeptide fragment” refers toa portion of a polypeptide that results from digestion of a polypeptidewith a protease. Unless otherwise specified, “a,” “an,” “the,” and “atleast one” are used interchangeably and mean one or more than one. Theterms “comprises” and variation thereof do not have a limiting mailingwhere these terms appear in the description and claims.

A polypeptide of the present invention may be characterized by molecularweight, mass fingerprint, or the combination thereof. The molecularweight of a polypeptide, typically expressed in kilodaltons (kDa), canbe determined using routine methods including, for instance, gelfiltration, gel electrophoresis including sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis (PAGE), capillary electrophoresis,mass spectrometry, and liquid, chromatography including HPLC.Preferably, molecular weight is determined by resolving a polypeptideusing an SDS polyacrylamide gel having a stacking gel of about 4% and aresolving gel of about 10% and/or reducing and denaturing conditions.Unless indicated otherwise, molecular weight refers to molecular weightas determined by SDS-PAGE. As used herein, a “mass fingerprint” refersto a population of polypeptide fragments obtained from a polypeptideafter digestion with protease. Typically, the polypeptide fragmentsresulting from a digestion are analysed using a mass spectrometricmethod. Each polypeptide fragment is characterized by a mass, or by amass (m) to charge (z) ratio, which is referred to as an “m/z ratio” oran “m/z value”. Methods for generating a mass fingerprint of apolypeptide are routine. An example of such a method is disclosed inExample 13.

Polypeptides of the present invention may be metal regulatedpolypeptides. As used herein, a “metal regulated polypeptide” is apolypeptide that is expressed by a microbe at a greater levels when themicrobe is grown in low metal conditions compared to growth of the samemicrobe in high metal conditions. Low metal and high metal conditionsare described herein. For instance, one class of metal regulatedpolypeptide produced by Staphylococcus spp. not expressed at detectablelevels during growth of the microbe in high metal conditions but isexpressed at detectable levels during growth in low metal conditions.Examples of such metal regulated polypeptides isolatable from S. aureusafter growth in low iron conditions have molecular weights of 88 kDa, 55kDa, 38 kDa, 37 kDa, 36 kDa, 35 kDa, and 33 kDa. Examples of such metalregulated polypeptides isolatable from S. aureus after growth in lowzinc or low copper conditions have molecular weights of 115 kDa, 88 kDa,80 kDa, 71 kDa, 69 kDa, 35 kDa, 30 kDa, 29 kDa, and 27 kDa.

The present invention also includes polypeptides that are not metalregulated. Such polypeptides are expressed in the presence of a metalion such as ferric chloride, and also expressed when grown in low ironconditions. Examples of such polypeptides isolatabie from S. aureus havemolecular weights of 150 kDa, 132 kDa, 120 kDa, 75 kDa, 58 kDa, 50 kDa,44 kDa, 43 kDa, 41 kDa, and 40 kDa.

Whether a polypeptide is a metal regulated polypeptide or not can bedetermined by methods useful for comparing the presence of polypeptides,including, for example, gel filtration, gel electrophoresis includingsodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE),capillary electrophoresis, mass spectrometry, and liquid chromatographyincluding HPLC. Separate cultures of a microbe are grown under highmetal conditions and under low metal conditions, polypeptides of thepresent invention are isolated as described herein, and the polypeptidespresent in each culture are resolved and compared. Typically, an equalamount of polypeptides from each culture is used. Preferably, thepolypeptides are resolved using an SDS polyacrylamide get having astacking gel of about 4% and a resolving gel of about 10% under reducingand denaturing conditions. For instance, 30 micrograms (μg) of totalpolypeptide from each culture may be used and loaded into wells of agel. After running the gel and staining the polypeptides with CoomasieBrilliant Blue, the two lanes can be compared. When determining whethera polypeptide is or is not expressed at a detectable level, 30 μg oftotal polypeptide from a culture is resolved on an SDS-PAGE gel andstained with Coomasie Brilliant Blue using methods knows in the art. Apolypeptide that can be visualised by eye is considered to be expressedat a detectable level, while a polypeptide that cannot be visualized byeye is considered to not be expressed at a detectable level.

Polypeptides of the present indention may have immunogenic activity.“Immunogenic activity” refers to the ability of a polypeptide to elicitan immunological response in an animal. An immunological response to apolypeptide is the development in an animal of a cellular and/orantibody-mediated immune response to the polypeptide. Usually, animmunological response includes but is not limited to one or more of thefollowing effects: the production of antibodies, B cells, helper Tcells, suppressor T cells, and/or cytotoxic T cells, directed to anepitope or epitopes of the polypeptide. “Epitope” refers to the site onan antigen to which specific B cells and/or T cells respond so thatantibody is produced. The immunogenic activity may be protective.“Protective immunogenic activity” refers to the ability of a polypeptideto elicit an immunological response in an animal that prevents orinhibits infection by Staphylococcus spp., for instance, S. aureus.Whether a polypeptide has protective immunogenic activity can bedetermined by methods known in the art, for instance as described isExamples 5, 9, or 12. For example, a polypeptide of the presentinvention, or combination of polypeptides of the present invention,protect a rodent such as a mouse against challenge with a Staphylococcusspp. A polypeptide of the present invention may have seroactiveactivity. “Seroactive activity” refers to the ability of a candidatepolypeptide to react with antibody present in convalescent serum from ananimal infected with a Staphylococcus spp., for instance, S. aureus. Insome aspects, the convalescent serum may be from an animal infected withthe ATCC isolate 19636, strain SAAV1, strain 2176, or strain 1477.Polypeptides of the present invention may have immunoregulatoryactivity. “Immunoregulatory activity” refers to the ability of apolypeptide to act in a nonspecific manner to enhance an immune responseto a particular antigen. Methods for determining whether a polypeptidehas immunoregulatory activity are known in the art.

A polypeptide of the present invention may have the characteristics of apolypeptide expressed by a reference microbe. The characteristics caninclude both molecular weight and mass fingerprint. The referencemicrobe can be a gram positive, preferably a member of the familyMicrococcaceae, preferably, Staphylococcus spp., more preferably,Staphylococcus aureus. Preferred examples of strain are detailed inTable 1.

TABLE 1 Bacterial strains. Bacterial cell Laboratory designation S.aureus ATCC isolate 19636 S. aureus strain SAAV1 S. aureus strain 1477S. aureus strain 2176

When the reference microbe is S. aureus ATCC isolate 19636, a candidatepolypeptide is considered to be a polypeptide of the present inventionif it has a molecular weight of 88 kDa, 55 kDa, 38 kDa, 37 kDa, 36 kDa35 kDa, or 33 kDa, and has a mass fingerprint that is similar to themass fingerprint of a metal regulated polypeptide expressed by areference microbe and having a molecular weight of 88 kDa, 55 kDa, 38kDa, 37 kDa, 36 kDa 35 kDa, or 33 kDa, respectively. Preferably, suchpolypeptides are metal regulated. For instance, a candidate polypeptideis a polypeptide of the present invention if it has a molecular weightof 88 kDa and has a mass fingerprint similar to the mass fingerprint ofan 88 kDa metal regulated polypeptide produced by the reference strainS. aureus ATCC isolate 19636.

When the reference microbe is S. aureus isolate SAAV1, a candidatepolypeptide is considered to be a polypeptide of the present inventionif it has a molecular weight (as determined by SDS-PAGE) of 88 kDa, 55kDa, 38 kDa, 37 kDa, 36 kDa, 35 kDa, or 33 kDa, and has a massfingerprint that is similar to the mass fingerprint of a polypeptideexpressed by a reference microbe and having a molecular weight (asdetermined by SDS-PAGE) of 88 kDa, 55 kDa, 38 kDa, 37 kDa, 36 kDa, 35kDa, or 33 kDa, respectively. Preferably, such polypeptides are metalregulated. For instance, a candidate polypeptide is a polypeptide of thepresent invention if it has a molecular weight of 88 kDa and has a massfingerprint similar to the mass fingerprint of an 88 kDa metal regulatedpolypeptide produced by the reference strain S. aureus isolate SAAV1.

When the reference microbe is S. aureus strain 2176, a candidatepolypeptide is considered to be a polypeptide of the present inventionif it has a molecular weight (as determined by SDS-PAGE) of 88 kDa, 80kDa, 65 kDa, 55 kDa, 37 kDa, 36 kDa, 35 kDa, 33 kDa, or 32 kDa, and hasa mass fingerprint that is similar to the mass fingerprint of apolypeptide expressed by a reference microbe and having a molecularweight (as determined by SDS-PAGE) of 88 kDa, 80 kDa, 65 kDa, 55 kDa, 37kDa, 36 kDa, 35 kDa, 33 kDa, or 32 kDa, respectively. Preferably, suchpolypeptides are metal regulated. For instance, a candidate polypeptideis a polypeptide of the present invention if it has a molecular weightof 88 kDa and has a mass fingerprint similar to the mass fingerprint ofan 88 kDa metal regulated polypeptide produced by the reference strainS. aureus isolate 2176.

When the reference microbe is S. aureus strain 1477, a candidatepolypeptide is considered to be a polypeptide of the present inventionif it has a molecular weight (as determined by SDS-PAGE) of 88 kDa, 80kDa, 65 kDa, 55 kDa, 37 kDa, 36 kDa, 35 kDa, 33 kDa, or 32 kDa, and hasa mass fingerprint that is similar to the mass fingerprint of apolypeptide expressed by a reference microbe and saving a molecularWeight (as determined by SDS-PAGE) of 88 kDa, 80 kDa, 65 kDa, 55 kDa, 37kDa, 36 kDa, 35 kDa, 33 kDa, or 32 kDa, respectively. Preferably, suchpolypeptides are metal regulated. For instance, a candidate polypeptideis a polypeptide of the present invention if it has a molecular weightof 88 kDa and has a mass fingerprint similar to the mass fingerprint ofan 88 kDa metal regulated polypeptide produced by the reference strainS. aureus isolate 1477.

The polypeptides expressed by a reference microbe and referred to aboveby molecular weight can be obtained by growth of the reference microbeunder low metal conditions and the subsequent isolation of a polypeptideby the processes disclosed herein. A candidate polypeptide is isolatablefrom a microbe, preferably a gram, positive microbe, more preferably, amember of the family Micrococcaceae, preferably, Staphylococcus spp.,more preferably, Staphylococcus aureus.

Other gram positive microbes from which polypeptides can be isolatedinclude Cornebacterium spp., Enterococcus spp., Erysipelothrix spp.,Kytococcus spp., and Micrococcus spp., Mycobacterium spp., andErysipelothrix spp. A candidate polypeptide may also be produced usingrecombinant, enzymatic, or chemical techniques.

A candidate polypeptide may be evaluated by mass spectrometry analysisto determine whether the candidate polypeptide has a mass fingerprintsimilar to one of the polypeptides expressed by a reference microbe andreferred to above by molecular weight. Typically, the candidatepolypeptide is isolated, for instance by resolving the candidatepolypeptide by gel electrophoresis and excising the portion of the gelcontaining the candidate polypeptide. Any gel electrophoresis methodthat separates polypeptides based on differing characteristics can beused, including 1 dimensional or 2 dimensional gel electrophoresis, aswell as liquid chromatographic separation based on, for instance,hydrophobicity, pI, or size. The candidate polypeptide is fragmented,for instance by digestion with a protease. Preferably, the proteasecleaves the peptide bond on the carboxy-terminal side of the amino acidlysine and the amino acid arginine, except when the amino acid followingthe lysine or the arginine is a proline. An example of such a proteaseis trypsin. Methods for digesting a polypeptide with trypsin are realiseand known in the art. An example of such a method is disclosed inExample 13.

Methods for the mass spectrometric analysis of polypeptides are routineand known in the art and include, but are not limited to, matrixassisted laser desorption/ionization time of flight mass spectroscopy(MALDI-TOF MS). Typically, a mixture containing the polypeptidefragments obtained from a candidate polypeptide is mixed with a matrixthat functions to transform the laser energy to the sample and produceionized, preferably monoisotopic, polypeptide fragments. Examples ofmatrices that can be used include, for instance, sinapinic acid orcyano-4-hydroxycinnamic acid. An sample of a method for the analysis ofpolypeptides by MALDI-TOF MS is described in Example 13. The ionizedpolypeptide fragments are separated according to their m/z ratio, anddetected to yield a spectrum of m/z ratio versus intensity. The spectrumincludes m/z values that represent the polypeptide fragments derivedfrom the candidate, polypeptide. For any givers polypeptide, the amountof each polypeptide fragment reaching from a trypsin digestion should beequimolar. However, it is known that trypsin digestion is not always100% efficient, for instance, some sites are more efficiently cleaved.Thus, when MALDI-TOF MS is used to determine m/z values, the intensityof each m/z value is typically not identical. Generally, a spectrum hasa background level of noise present across most of the x-axis (i.e., theaxis having the values of the m/z ratios). This background level ofnoise varies depending on the running conditions and the machine used,and is easily identified by visual inspection of the spectrum. An m/zvalue is generally considered to represent a polypeptide fragment whenthe intensity is at least 2 times greater, at least 3 times greater, orat least 4 times greater than the background level of noise. Thespectrum usually includes other m/z values that are artifacts resultingfrom, for instance, incomplete digestion, over digestion, otherpolypeptides that may be present in the mixture, or the protease used todigest the polypeptide including m/z values resulting from autolysis ofthe protease. This method of digesting a polypeptide with a protease isrecognized in the art as resulting in a mass fingerprint of greatspecificity that can be used to accurately characterize the polypeptideand distinguish it from other polypeptides.

In this aspect of the invention, when a candidate polypeptide isanalyzed by mass spectroscopy, preferably both the candidate polypeptideand the polypeptide from the reference microbe are prepared and analyzedtogether, thereby decreasing any potential artifacts resulting fromdifferences in sample handling and running conditions. Preferably, allreagents used to prepare and analyse the two polypeptides are the same.For instance, the polypeptide from the reference microbe and thecandidate polypeptide are isolated under substantially the sameconditions, fragmented under substantially the same conditions, andanalyzed by MALDI-TOF MS on the same machine under substantially thesame conditions. A mass fingerprint of a candidate polypeptide isconsidered to be similar to the mass fingerprint of a polypeptide from areference microbe when at least 80%, at least 90%, at least 95%, orsubstantially all of the m/z values present in the spectrum of thereference microbe polypeptide and above the background level of noiseare also present in the spectrum of the candidate polypeptide.

In another aspect, a polypeptide is considered to be a polypeptide ofthe present invention if it has a molecular weight of a referencepolypeptide described in Table 2, 3, 4, or 5 and has a mass fingerprintthat includes the population of polypeptide fragments of the referencepolypeptide as listed in Table 2, 3, 4, or 5. For instance, apolypeptide of the present invention includes a polypeptide of 88 kDaand a mass fingerprint that includes polypeptide fragments having massesof HVDVR (SEQ ID NO: 1), YSYER (SEQ ID NO: 2), IIGDYRR (SEQ ID NO: 3),IFTDYRK (SEQ ID NO: 4), ELKELGQK (SEQ ID NO: 5), YAQVKPIR (SEQ ID NO:6), QMQFFGAR (SEQ ID NO: 7), SMQPFGGIR (SEQ ID NO: 8), VSGYAVNFIK (SEQID NO: 9, NHATAWQGFK (SEQ ID NO: 10), LWEQVMQLSK (SEQ ID NO: 11),SLGKEPEDQNR (SEQ ID NO: 12), DGISNTFSIVPK (SEQ ID NO: 13), AGVITGLPDAYGR(SEQ ID NO: 14), TSTFLDIYAER (SEQ ID NO: 15), SMQPFGGIRMAK (SEQ ID NO:16), THNQGVFDAYSR (SEQ ID NO: 17), KAGVITGLPDAYGR (SEQ ID NO: 18),TLLYAINGGKDEK (SEQ ID NO: 19), IEMALHDTEIVR (SEQ ID NO: 20),AGEPFAPGANPMHGR (SEQ ID NO: 21), VALYGVDFLMEEK (SEQ ID NO: 22),KTHNQGVFDAYSR (SEQ ID NO: 23), YGFDLSRPAENFK (SEQ ID NO: 24),TSSIQYENDDIMR (SEQ ID NO: 25), KAGEPFAPGANPMHGR (SEQ ID NO: 26),RVALYGVDFLMEEK (SEQ ID NO: 27), LWEQVMQLSKEER (SEQ ID NO: 28),MLETNKNHATAWQGFK (SEQ ID NO: 29), MHDFNTMSTEMSEDVIR (SEQ ID NO: 30),YGNNDDRVDDIAVDLVER (SEQ ID NO: 31), ETLIDAMEHPEEYPQLTIR (SEQ ID NO: 32),YAQVKPIRNEEGLVVDFEIEGDFPK (SEQ ID NO: 33). The mass fingerprint of acandidate polypeptide can be determined by a mass spectrometric method,for instance by MALDI-TOF MS. The mass fingerprint of a candidatepolypeptide will generally have additional polypeptide fragments andtherefore additional m/z values other than those listed for apolypeptide in Table 2, 3, 4, or 5. Preferably, when the candidatepolypeptide is being compared to a polypeptide in Table 2, 3, 4, or 5,the candidate polypeptide is isolatable from a microbe, preferably agram positive microbe, more preferably, a member of the familyMicrococcaceae, preferably, Staphylococcus spp., more preferably,Staphylococcus aureus. Other gram positive microbes includeCorynebacterium spp., Enterococcus spp., Erysipelothrix spp., Kytococcusspp., Listeria spp., Micrococcus spp., and Mycobacterium spp., andErysipelothrix spp. A candidate polypeptide can be obtained by growth ofa microbe under low metal conditions and the subsequent isolation of apolypeptide by the processes described herein.

If is well known in the art that modifications of amino acids can beaccidentally introduced during sample handling, such as oxidation, andformation of carbamidomethyl derivatives. Further, thus types ofmodifications alter the m/z value of a polypeptide fragment. Forinstance, if a polypeptide fragment contains a methionine that isoxidized, the m/z value will be increased by 16 relative to the samefragment that does not contain the oxidized methionine. Accordingly,those polypeptide fragments in Tables 2, 3, 4, or 5 having the notation“oxidation (M)” have an m/z value that is increased by 16 relative tothe same fragment that does not contain the oxidized methionine. It isunderstood that the polypeptide fragments of Table 2, 3, 4, or 5 can bemodified during sample handling.

TABLE 2 Characteristics of polypeptides obtained from S. aureus ATCC isolate 19636. Approximate m/z value ofmolecular polypeptide weight in fragments Polypeptide kilodaltonsresulting from Predicted amino acid sequence  SEQ  designation (kDa)¹trypsin digest² of the polypeptide fragment ID NO: P23 88 625.4 HVDVR  1 717.3 YSYER   2 892.5 IIGDYRR   3 942.5 IFTDYRK   4 944.5 ELKELGQK  5 974.6 YAQVKPIR   6 984.5 QMQFFGAR   7 992.5 SMQPFGGIR   8 1097.6VSGYAVNFIK   9 1159.5 NHATAWQGFK  10 1261.7 LWEQVMQLSK  11 1272.7SLGKEPEDQNR  12 1277.7 DGISNTFSIVPK  13 1289.7 AGVITGLPDAYGR  14 1315.7TSTFLDIYAER  15 1322.7 SMQPFGGIRMAK  16 1394.7 THNQGVFDAYSR  17 1417.8KAGVITGLPDAYGR  18 1421.8 TLLYAINGGKDEK  19 1426.8 IEMALHDTEIVR  201508.8 AGEPFAPGANPMHGR  21 1513.8 VALYGVDFLMEEK  22 1522.8 KTHNQGVPDAYSR 23 1543.8 YGFDLSRPAENFK  24 1571.8 TSSIQYENDDIMR  25 1636.9KEGEPFAPGANPMHGR  26 1670.0 RVALYGVDFLMEEK  27 1676.0 LWEQVMQLSKEER  281876.2 MLENTNKNHATAWQGFK  29 2043.1 MHDFNTMSTEMSEDVIR  30 2078.2YGNNDDRVDDIAVDLVER  31 2285.5 ETLIDAMEHPEEYPQLTIR  32 2892.9YAQVKPIRNEEGLVVDFEIEGDFPK  33 P25 55 783.6 LHSWLK  34 911.7 KLHSWLK  35937.6 TYTFHLR  36 996.6 KFDGTGPFK  37 1025.6 QAIGHMVNR  38 1063.6KWDVSEDGK  39 1185.6 IYNSIDDAFK  40 1277.6 NLEMAMYYDK  41 1324.7ENKQLTYTTVK  42 1346.7 AESLLDEAGWKK  43 1381.8 TVRQAIGHMVNR  44 1394.8TYTFHLRDDVK  45 1400.7 KGETNFAFTDDR  46 1419.7 FHDGTPFDADAVK  47 1422.8NVTDINFDMPTR  48 1428.8 DKIYNSIDDAFK  49 1483.8 EQAEYLQAEFKK  50 1509.8VMPAGETAFLSMKK  51 1547.9 FHDGTPFDADAVKK  52 1550.9 NVTDINFDMPTRK  531559.9 LNINGETSDKIAER  54 1788.1 EILDGQEKPATQLFAK  55 1930.1GSSSQKEQAEYLQAEFK  56 1946.0 DESADFNKNDQYWGEK  57 2100.4IAKEILDGQEKPATQLFAK  58 2239.3 VSFTQSQYELPFNEMQYK  59 2493.5EAYQPALAELAMPRPYVFVSPK +  60 Oxidation (M) 2900.6DIGDMNPHVYGGSMSAESMIYEPLVR +  61 2 Oxidation (M) 2916.6DIGDMNPHVYGGSMSEESMIYEPLVR +  62 3 Oxidation (M) P26 38 993.6 IVYVGADEK 63 996.7 QALNNPVLK  64 1237.7 ETVKIENNYK  65 1272.7 ENPDVILAMDR  661502.0 IAATKPEVIFISGR  67 1507.9 NAVVLDYGALDVMK  68 1523.9 ALPNFLESFKDDK 69 1559.9 LWYFAAGSTTTTIK  70 1716.0 FGGLVYDTLGFNAVDK  71 1737.0IVYVGADEKNLIGSMK  72 1844.1 FGGLVYDTLGFNAVDKK  73 1929.1GRFGGLVYDTLGFNAVDK  74 1998.2 TVMYLLVNEGELSTFGPK  75 2234.4EVNFDKIAATKPEVIFISGR  76 3143.8 VSNSNHGQNVSNEYVNKENPDVILAMDR  77 P27 37699.5 FEYIK  78 729.4 DAWPLK  79 792.5 ASVVNFR  80 852.4 VYDQLSK  81987.5 HAMGTTEIK  82 1008.5 LIDDLYEK  83 1020.5 YKDAWPLK  84 1074.5EKEAEDLLK  85 1083.6 LKPDLIVASK  86 1169.5 FEYIKNDLK  87 1182.5KTESEWTSSK  88 1184.5 YDDKVAAFQK  89 1223.5 NEKVYDQLSK  90 1278.6IAPTVSTDTVFK  91 1497.6 TESEWTSSKEWK  92 1502.7 DAWPLKASVVNFR  93 1558.8QVDNGKDIIQLTSK  94 1605.8 LIDDLYEKLNIEK  95 1623.8 IVGQEPAPNLEEISK  961712.8 ESIPLMNADHIFVVK  97 1800.9 IYAGGYAGEILNDLGFK  98 1957.0IYAGGYAGEILNDLGFKR  99 2252.0 NNQVSDDLDEITWNLAGGYK 100 3383.9RVVTLYQGATDVAVSLGVKPVGAVESWTQKPK 101 P28 36 646.4 DVWAR 102 725.5 IIKPVR103 1068.4 IGDYTSVGTR 104 1185.5 KQPNLEEISK 105 1327.6 LKPDLIIADSSR 1061343.6 VDIVDRDVWAR 107 2080.9 GPYLQLDTEHLADLNPER 108 2438.1AGLLAHPNYSYVQGFLNELGFK 109 2789.4 IVVLEYSFADALAALDVKPVGIADDGK 110 P29 35760.5 AGWAEVK 111 1012.6 TVDIPKDPK 112 1107.6 KDWEETTAK 113 1204.7VAPTVVVDYNK 114 1238.6 YLEQQEMLGK 115 1244.6 LYTYGDNWGR 116 1259.7IAVVAPTYAGGLK 117 1281.7 GGEVLYQAFGLK 118 1516.8 AGWAEVKQEEIEK 1191683.9 LGANIVAVNQQVDQSK 120 1877.1 EKPDLIIVYSTDKDIK 121 1884.0AIGQDATVSLFDEFDKK 122 2227.1 VDAGTYWYNDPYTLDFMR 123 2781.4YAGDYIVSTSEGKPTPGYESTNMWK 124 P30 33 834.5 QAIEFVK 125 864.5 YIAQLEK 126946.5 QGTPEQMR 127 962.5 QAIEFVKK 128 976.5 DKFNDIPK 129 1054.5AMITSEGAFK 130 1202.5 SNIETVHGSMK 131 1268.6 HLLVETSVDKK 132 1443.6DIFGEVYTDSIGK 133 1450.7 TIQQTFIDNDKK 134 1454.7 VVTTNSILYDMAK 1351571.7 KDIFGEVYTDSIGK 136 1593.7 QDPHAWLSLDNGIK 137 1818.9DVKPIYLNGEEGNKDK 138 1836.9 DKQDPHAWLSLDNGIK 139 1911.9 QYGITPGYIWEINTEK140 2582.3 LTDADVILYNGLNLETGNGWFEK 141 2710.2 KLTDADVILYNGLNLETGNGWFEK142 3942.4 NVGGDNVDIHSIVFVGQDPHEYEVKPK 143 ¹Molecular weight asdetermined by SDS-PAGE. ²The m/z value of a polypeptide fragment can beconverted to mass by subtracting 1 from the m/z value. Each massincludes a range of plus or minus 300 parts per million (ppm), or plusor minus 1 Da.

TABLE 3 Characteristics of polypeptides obatined from S. aureus isolate SAVV1. Approximate m/z value ofmolecular polypeptide weight in fragments polypeptide kilodaltonsresulting from Predicted amino acid sequence SEQ designation (kDa)¹trypsin digest² of the polypeptide fragment ID NO: P33A 55 783.4 LHSWLK144 911.5 KLHSWLK 145 937.5 TYTFHLR 146 996.5 KFDGTGPFK 147 1025.5QAIGHMVNR 148 1039.4 NDQYWGEK 149 1178.5 GTDSLDKDSLK 150 1185.5IYNSIDDAFK 151 1222.6 DKYTVELNLK 152 1229.5 ISTLIDNVKVK 153 1346.6AESLLDEAGWKK 154 1355.5 EQAEYLQAEFK 155 1381.6 VMPAGETAFLSMK 156 1400.5KGETNFAFTDDR 157 1419.6 FHDGTPFDADAVK 158 1422.6 NVTDINFDMPTR 159 1483.6EQAEYLQAEFKK 160 1547.7 FHDGTPFDADAVKK 161 1550.6 NVTDINFDMPTRK 1621559.7 LNINGETSDKIAER 163 1787.9 EILDGQEKPATQLFAK 164 1945.8DESADFNKNDQYWGEK 165 2239.0 VSFTQSQYELPFNEMQYK 166 2354.1QIDDEGIFIPISHGSMTVVAPK 168 2868.1 DIGDMNPHVYGGSMSAESMIYEPLVR 169 P33B 55895.4 FPYAANGR 170 904.5 ALLHASNR 171 1045.5 EEGLAIKASK 172 1384.5GEAYFVDNNSLR 173 1435.7 TIEADYVLVTVGR 174 1669.8 RPNTDELGLEELGVK 1751841.0 NAIIATGSRFIEIPNFK 176 2179.2 TSISNIYAIGDIVPGLPLAHK 177 2546.2FVEAQHSENLGVIAESVSLNFQK 178 2587.3 VVGDFPIETDTIVIGAGPGGYVAAIR 179 P35 37699.4 FEYIK 180 729.4 DAWPLK 181 792.4 ASVVNFR 182 852.4 VYDQLSK 1831008.4 LIDDLYEK 184 1020.4 YKDAWPLK 185 1074.4 EKEAEDLLK 186 1083.5LKPDLIVASK 187 1169.5 FEYIKNDLK 188 1182.4 KTESEWTSSK 189 1184.4YDDKVAAFQK 190 1278.5 IAPTVSTDTVFK 191 1558.7 QVDNGKDIIQLTSK 192 1623.7IVGQEPAPNLEEISK 193 1712.7 ESIPLMNADHIFVVK 194 1800.7 IYAGGYAGEILNDLGFK195 1956.8 IYAGGYAGEILNDLGFKR 196 2251.9 NNQVSDDLDEITWNLAGGYK 197 3227.5VVTLYQAGTDVAVSLGVKPVGAVESWTQKPK 198 P38 33 864.5 YIAQLEK 199 946.4QGTPEQMR 200 976.5 DKFNDIPK 201 1054.5 AMITSEGAFK 202 1146.5 FNDIPKEQR203 1268.6 HLLVETSVDKK 204 1322.5 TIQQTFIDNDK 205 1443.6 DIFGEVYTDSIGK206 1450.6 TIQQTFIDNDKK 207 1454.6 VVTTNSILYDMAK 208 1593.7QDPHAWLSLDNGIK 209 1818.9 DVKPIYLNGEEGNKDK 210 1836.8 DKQDPHAWLSLDNGIK211 1911.9 QYGITPGYIWEINTEK 212 2942.4 NVGGDNVDIHSIVPVGQDPHEYEVKPK 213¹Molecular weight as determined by SDS-PAGE. ²The m/z value of apolypeptide fragment can be converted to mass by subtracting 1 from them/z value. Each mass includes a range of plus or minus 300 parts permillion (ppm), or plus or minus 1 Da.

TABLE 4 Characteristics of polypeptides obatined from S. aureus isolate 2176. Approximate m/z value of molecularpolypeptide weight in fragments Polypeptide kilodaltons resulting fromPredicted amino acid sequence SEQ designation (kDa)¹ trypsin digest²of the polypeptide fragment ID NO: P478 88 736.35 IIGDYR 213 814.49IFTDYR 214 942.42 IFTDYRK   4 945.36 TGNTPDGRK 215 974.40 YAQVKPIR   6984.27 QMQFFGAR   7 992.41 SMQPFGGIR   8 1087.31 EQQLDVISR 216 1097.31VSGYAVNFIK   9 1159.37 NHATAWQGFK  10 1261.37 LWEQVMQLSK  11 1289.46AGVITGLPDAYGR  14 1315.42 TSTFLDIYAER  15 1322.39 LREELSEQYR 217 1394.37THNQGVFDAYSR  17 1417.52 KAGVITGLPDAYGR  18 1426.36 IEMALHDTEIVR  201487.39 NHATAWQGFKNGR 218 1508.42 AGEPFAPGANPMHGR  21 1513.52VALYGVDFLMEEK  22 1543.43 YGFDLSRPAENFK  24 1571.50 TSSIQYENDDIMR  251636.56 KAGEPFAPGANPMHGR  26 1859.80 DLETIVGVQTEKPFKR 219 1876.77TMATGIAGLSVAADSLSAIK 220 2042.57 MHDFNTMSTEMSEDVIR  30 2077.68YGNNDDRVDDIAVDLVER  31 2158.88 AGVITESEVQEIIDHFIMK 221 2284.90ETLIDAMEHPEEYPQLTIR  32 2575.08 FLHSLDNLGPAPEPNLTVLWSVR 222 2628.01SGAQVGPNFEGINSEVLEYDEVFK 223 2756.06 SGAQVGPNFEGINSEVLEYDEVFKK 2243262.33 VASTITSHDAGYLDKDLETIVGVQTEKPFK 225 P479 80 625.27 HVDVR   1736.26 IIGDYR 226 814.22 IFTDYR 227 942.27 IFTDYRK   4 974.26 YAQVKPIR  6 984.18 QMQFFGAR   7 992.23 SMQPFGGIR    8 1087.16 EQQLDVISR 2281097.24 VSGYAVNFIK   9 1159.12 NHATAWQGFK  10 1243.14 VDDIAVDLVER 2291261.22 LWEQVMQLSK  11 1272.24 SLGKEPEDQNR  12 1277.18 DGISNTFSIVPK  131289.21 AGVITGLPDAYGR  14 1315.19 TSTFLDIYAER  15 1322.21 LREELSEQYR 2301394.16 THNQGVFDAYSR  17 1417.32 KAGVITGLPDAYGR  18 1426.23 IEMALHDTEIVR 20 1487.19 NHATAWQGFKNGR 231 1508.25 AGEFFAPGANPMHGR  21 1513.21VALYGVDFLMEEK  22 1522.25 KTHNQGVFDAYSR  23 1543.26 YGFDLSRPAENFK  241571.23 TSSIQYENDDIMR  25 1636.29 KAGEPFAPGANPMHGR  26 1703.43DLETIVGVQTEKPFK 232 1751.45 EAVQWLYAYLAAIK 233 1859.53 DLETIVGVQTEKPFKR234 1876.50 TMATGIAGLSVAADSLSAIK 235 1936.37 NEEGLVVDFEIEGDFPK 2362042.43 MHDFNTMTSTEMSEDVIR  30 2077.45 YGNNDDRVDDIAVDLVER  31 2158.57AGVITESEVQEIIDHFIMK 237 2284.61 ETLIDAMEHPEEYPQLTIR  32 2574.77FLHSLDNLGPAPEPNLTVLWSVR 238 2627.61 SGAQVGPNFEGINSEVLEYDEVFK 239 2755.70SGAQVGPNFEGINSEVLEYDEVFKK 240 2907.65 EFIQLNYTLYEGNDSFLAGPTEATSK 2413261.91 VASTITSHDAGYLDKDLETIVGVQTEKPFK 242 3421.02TPDYNELFSGDPTWVTESIGGVGIDGRPLVTK 243 P480 65 625.35 HVDVR   1 717.38YSYER   2 733.42 LPDNFK 244 736.44 IIGDYR 245 814.33 IFTDYR 246 853.31YGNNDDR 247 842.33 IFTDYRK   4 844.39 ELEKLGQK   5 974.52 YAQVKPIR   6984.36 QMQFFGAR   7 992.44 SMQPFGGIR   8 1049.44 TLLYAINGGK 248 1087.43EQQLDVISR 249 1097.51 VSGYAVNFIK   9 1159.52 NHATAWQGFK  10 1289.53AGVITGLPDAYGR  14 1315.51 TSTFLDIYAER  15 1322.46 LREELSEQYR 250 1394.50THNQGVFDAYSR  17 1417.65 KAGVITGLPDAYGR  18 1442.56 IEMALHDTEIVR +Oxidation (M) 251 1467.60 VSGYAVNFIKLTR 252 1522.61 KTHNQGVFDAYSR  231524.55 AGEPFAPGANPMHGR + Oxidation  (M) 253 1529.64 VALYGVDFLMEEK +Oxidation  (M) 254 1543.62 YGFDLSRPAENFK  24 1652.68 KAGEPFAPGANPMHGR +Oxidation  (M) 255 1671.76 TSTFLDIYAERDLK 256 1766.76 VDDIAVDLVERFMTK +Oxidation  (M) 257 1876.86 TMATGIAGLSVAADSLSAIK 258 2077.93YGNNDDRVDDIAVDLVER  31 2225.07 DSEHTMSVLTITSNVVYGKK + Oxidation  (M) 2592575.33 FLHSLDNLGPAPEPNLTVLWSVR 260 2628.25 SGAQVGPNFEGINSEVLEYDEVFK 2612748.36 NLTSMLDGYAMQCGHHLNINVFNR 262 2756.63 SGAQYGPNFEGINSEVLEYDEVFKK263 3001.02 DEKSGAQVGPNFEGINSEVLEYDEVFK 263 3420.75TPDYNELFSGDPTWVTESIGGVGIDGRPLVTK 265 P481 55 634.33 AKSNSK 266 883.24TFYPEAR 267 1014.24 QFWGHLVK 268 1131.17 WIPLMMKGR 269 1207.21VINEEFEISK 270 1324.10 NEDWQLYTAGK 271 1360.28 TLLFGPPANVGPK 272 1386.31LDRFAIESSNER 273 1565.30 IDEGTDVNFGELTR 274 1584.34 EFINPLFHISYVR 2751699.29 EIEPDWNIHVYER 276 1744.36 EPPGTPPMTVPHLDTR 277 2046.52QVTDYVFIGAGGGAIPLLQK 278 2189.43 TFYPEARNEDWQLYTAGK 279 2806.58HLGGFPISGQFLACTNPQVIEQHDAK 280 P482 37 699.28 FEYIK 281 729.26 DAWPLK282 792.33 ASVVNFR 283 852.28 VYDQLSK 284 1008.30 LIDDLYEK 285 1020.31YKDAWFLK 286 1083.43 LKPDLIVASK 287 1278.36 IAFTVSTDTVFK 288 1623.44IVGQEPAPNLEEISK 289 1712.62 ESIPLMNADHIFVVK 290 1800.61 IYAGGAGEILNDLGFP291 1956.77 IYAGGYAGEILNDLGFKR 292 2251.77 NNQVSDDLDEITWNLAGGYK 2933227.44 VVTLYQGATDVAVSLGVKPVGAVESWTQKPK 294 P483 38 646.50 DVWAR 295672.41 KLNAVK 296 716.41 VDIVDR 297 725.61 IIKPVR 298 842.50 IAPTLSLK299 850.47 QNINSFK 300 1068.50 IGDYTSVGTR 301 1075.42 MIIMTDHAK +Oxidation  (M) 302 1185.53 KQPNLEEISK 303 1327.59 LKPDLIIADSSR 3041343.58 VDIVDRDVWAR 305 1592.76 LKPDLIIADSSRHK 306 2081.00GPYLQLDTEHLADLNPER 307 2438.24 AGLLAHPNYSYVGQGLNELGFK 308 2789.48IVVLEYSFADALAALDVKPGIADDGK 309 2917.60 IVVLEYSFADALAALDVKPVGIADDGKK 310P484 35 857.38 AAAIDLAGR 311 1022.23 NIEADTGMR + Oxidation  (M) 3121056.32 VVDANIAAQR 313 1075.36 ADIDLPFER 314 1285.44 LVGGAGEETIIAR 3151435.44 AMAVATEQEMKAR 316 1632.50 HHTEVLENPDNISK 317 1813.65VVEAESEVPLAMAEALR 318 1887.67 VIETPFIAGVAMNGIEVK 319 2299.85AGLALTTNQLESHYLAGGNVDR 320 2806.95 TVLSKGLDSGTAFEILSIDIADVDISK 3213337.42 AGLATTNQLESHYLAGGNVDRVVDANIAAQR 322 P485 33 625.28 ADYEK 323864.28 YIAQLEK 324 946.23 QGTPEQMR 325 1045.26 ALEQAGKSLK 326 1268.35JLLVETSVDKK 327 1443.34 DIFGEVYTDSIGK 328 1450.40 TIQQTFIDNDKK 3291454.37 VVTTNSILYDMAK 330 1571.45 KDIFGEVYTDSIGK 331 1576.44DVKPIYLNGEEGNK 332 1593.47 QDPHAWLSLDNGIK 333 1819.59 DVKPIYLNGEEGNKDK334 1836.62 DKQDPHAWLSLDNGIK 335 1911.66 QYGITPGYIWEINTEK 336 2172.83VIAVSKDVKPIYLNGEEGKN 337 2582.00 LTDADVILYNGLNETGNGWFEK 338 2942.26NVGGDNVDIHSIVPVGQDPHEYEVKPK 339 P486 32 625.42 ADYEK 340 864.41 YIAQLEK341 1268.48 HLLVETSVDKK 342 1443.49 DIFGEVYTDSIGK 343 1450.53TIQQTFIDNDKK 344 1454.61 VVTTNSILYDMAK 345 1576.64 DVKPIYLNGEEGNK 3461593.57 QDPHAWLSLDNGIK 347 1818.77 DVKPIYLNGEEGNKDK 348 1836.78DKQDPHAWLSLDNGIK 349 1911.81 QYGITPGYIWEINTEK 350 2582.18LTDADVILYNGLNLETGNGWFEK 351 2942.32 NVGGDNVDIHSIVPVGQDPHEYEVKPK 352¹Molecular weight as determined by SDS-PAGE. ²The m/z value of apolypeptide fragment can be converted to mass by subtracting 1 from them/z value. Each mass includes a range of plus or minus 400 parts permillion (ppm) or 1 Dalton.

TABLE 5Characteristics of polypeptides obatined from S. aureus isolate 1477.Approximate m/z value of molecular polypeptide weight in fragmentspolypeptide kilodaltons resulting from Predicted amino acid sequence SEQ designation (kDa)¹ trypsin digest² of the polypeptide fragmentID NO: P487 88 717.39 YSYER   2 736.52 IIGDYR 353 814.46 IFTDYR   354942.46 IFTDYRK   4 974.54 YAKVKPIR   6 984.41 QMQFFGAR   7 992.40SMQPFGGIR 8 1087.49 EQQLDVISR   355 1097.50 VSGYAVNFIK  9 1159.39NHATAWQGFK  10 1261.45 LWEQVMQLSK  11 1272.50 SLGKEPEDQNR  12 1277.50DGISNTFSIVPK  13 1289.54 AGVITGLPDAYGR  14 1315.54 TSTFLDIYAER 151322.53 LREELSEQYR 356 1394.50 THNQGVFDAYSR  17 1417.62 KAGVITGLPDAYGR 18 1426.65 IEMALHDTEIVR  20 1508.59 AGEPFAPGANPMHGR  21 1522.61KTHNQGVFDAYSR  23 1543.68 YGFDLSRPAENFK 24 1877.74 TMATGIAGLSVAADSLSAIK 357 2077.86 YGNNDDVRDDIAVDLVER 31 2159.08 AGVITESEVQEIIDHFIMK  3582285.07 ETLIDAMEHPEEYPQLTIR 32 2575.32 FLHSLDNLGPAPEPNLTVLWSVR 3592628.24 SGAQVGPNFEGINSEVLEYDEVFK 360 2756.41 SGAQVGPNFEGINSEVLEYDEVFKK361 3262.68 VASTITSHDAGYLDKDLETIVGVQTEKPFK   362 P488 80 625.49 HVDVR 1814.54 IFTDYR   363 842.66 IFTDYRK  4 974.69 YAQVKPIR   6 984.59QMQFFGAR   7 992.55 SMQPFGGIR  8 1159.64 NHATAWQGFK  10 1261.63LWEQVMQLSK  11 1272.74 SLGKEPEDQNR  12 1277.69 DGISNTFSIVPK  13 1289.76AGVITGLPDAYGR  14 1315.73 TSTFLDIYAER  15 1322.72 SMQPFGGIRMAK  161394.73 THNQGVFDAYSR  17 1417.86 KAGVITGLPDAYGR  18 1422.76TLLYAINGGKDEK  19 1426.80 IEMALHDETEIVR  20 1508.82 AGEPFAPGANFMHGR  211513.80 VALYGVDFLMEEK  22 1543.82 YGFDLSRPAENFK  24 1571.82TSSIQYENDDIMR 25 1703.99 DLETIVGVQTEKPFK 364 1860.23 DLETIVGVQTEKPFKR365 1877.07 TMATGIAGLSVAADSLSAIK 366 1937.09 NEEGLVVDFEIEGDFPK  3672078.13 YGNNDDRVDDIAVDLVER 31 2575.56 FLHSLDNLGPAPEPNLTVLWSVR 3682628.30 SGAQVGPNFEGINSEVLEYDEVFK 369 2908.63 EFIQLNYTLYEGNDSFLAGPTEATSK370 P489 65 733.67 IVKFAR   371 944.71 ELKELGQK   5 974.79 YAQVKPIR   6984.69 QMQFFGAR 7 1049.83 TLLYAINGGK 372 1087.78 EQQLDVISR   373 1097.79VSGYAVNFIR 9 1243.80 VDDIAVDLVER  374 1272.82 SLGKEPEDQNR  12 1289.87AGVITGLPDAYGR 14 1299.92 LPDNFKTYCAK  375 1315.83 TSTFLDIYAER  151322.84 SMQPFGGIRMAK 16 1390.93 DQKGALSSLSSVAK  376 1394.84 THNQGVFDAYSR17 1577.94 VASTITSHDAGYLDK  377 1637.09 KAGEPFAPGANPMHGR 26 1704.16DLETIVGVQTEKPFK 378 2030.42 MSIKTSSIQYENDDIMR  379 2078.34YGNNDDRVDDIAVDLVER  31 2284.60 ETLIDAMEHPEEYPQLTIR 32 2575.77FLHSLDNLGPAPEPNLTVLWSVR 380 2628.64 SGAQVGPNFEGINSEVLEYDEVFK 381 P490 55883.81 TFYPEAR 382 1014.87 QFWGHLVK 383 1131.97 WIPLMMKGR 384 1207.99VINEEFEISK 385 1231.97 YSFDQVIMTK 386 1325.02 NEDWQLYTAGK 387 1361.17TLLFGPFANVGPK 388 1362.14 GREDNPGIMAASK + Oxidation  (M) 389 1387.14LDRPAIESSNER 390 1481.24 NEDWQLYTAGKR 391 1566.28 IDEGTDVNFGELTR 3921585.34 EFINPLPHISYVR 393 1700.36 EIEPDWNIHVYER 394 1761.49EPPGTPPMTVPHLDTR + Oxidation  (M) 395 2047.67 QVTDYVFIGAGGGAIPLLQK 3962208.82 VYGKEPPGTPPMTVPHLDTR + Oxidation  (M) 397 2865.21HLGGFPISGQFLACTNPQVIEQHDAD 398 P492 36 857.57 AAAIDLAGR 399 1056.59VVDANIAAQR 400 1075.61 ADIDLPFER 401 1285.74 LVGGAGEETIIAR 402 1632.95HHTEVLENPDNISK 103 1814.09 VVEAESEVPLAMAEALR 404 2284.45AAAIDLAGRDVLEAVQMSVNPK + Oxidation  (M) 405 1300.40AGLALTTNQLESHYLAGGNVDR 406 2807.80 TVLSKGLDSGTAFEILSIDIADVDISK 407 P49335 762.46 FVFHGR 408 964.39 DGFNNIER 409 1363.56 GHVYNGISGGQFK 4101443.56 YTPTSILYFNPK 411 1450.64 QLAEDLQKHLGAK 412 1819.88NHSEYVTDMRLIGIR + Oxidation  (M) 413 1875.84 DLPPMEQVFDTLDLDK 4141941.00 IRPEDMHIMANIFLPK + Oxidation  (M) 415 2081.10 RIRPEDMHIMANIFLPK416 2283.30 ISHLVLTRTGLYIIDSQLLK 417 P495 32 ¹Molecular weight asdetermined by SDS-PAGE. ²The m/z value of a polypeptide fragment can beconverted to mass by subtracting 1 from the m/z value. Each massincludes a range of plus or minus 430 parts per million (ppm) or 1Dalton.

In yet archer aspect, the present invention further includespolypeptides having similarity with an amino acid sequence. Thesimilarity is referred to as structural similarity and is generallydetermined by aligning the residues of the two amino acid sequences(i.e., a candidate amino acid sequence and a reference amino acidsequence) to optimize the number of identical amino acids along thelengths of their sequences; gaps in either or both sequences arepermitted in making the alignment in order to optimize the number oridentical amino acids, although the amino acids in each sequence mustnonetheless remain in their proper order. Reference amino acid sequencesare disclosed in Tables 6, 7, 8, and 9. Two amino acid sequences can becompared using commercially available algorithms. Preferably, two aminoacid sequences are compared using the BLASTP program of the BLAST 2search algorithm, as described by Tatusova, et al., (FEMS Microbiol Lett1999, 174:247-250), and available through the World Wide Web, forinstance at the internet site maintained by the National Center forBiotechnology Information, National Institutes of Health. Preferably,the default values for all BLAST 2 search parameters are used, includingmatrix=BLOSUM62; open gap penalty=11, extension gap penalty=1, gapx_dropoff=50, expect=10, wordsize=3, and optionally, filter on. In thecomparison of two amino acid sequences using the BLAST search algorithm,structural similarity is referred to as “identities.” Preferably, acandidate amino acid sequence has at least 80% identity, at least 90%identity, at least 95% identity, at least 96% identity, at least 97%identity, at least 98% identity, or at least 99% identity to a referenceamino acid sequence. Preferably, the molecular weight of the candidateamino acid sequence and the reference amino acid sequence aresubstantially the same value. Preferably, the molecular weight of thecandidate amino acid sequence and the reference amino acid sequence isdetermined by SDS polyacrylamide gel electrophoresis. A candidatepolypeptide can be obtained by growth of a microbe under low metalconditions and the subsequent isolation of a polypeptide by theprocedures disclosed herein.

Typically, a candidate amino acid sequence having structural similarityto a inference amino acid sequence has immunogenic activity, protectiveimmunogenic activity, seroactive activity, immunoregulatory activity, ora combination thereof.

TABLE 6 S. aureus ATCC isolate 19636. NCBI sequence identifier ofpolypeptide identified by the Molecular weight of computer algorithm ashaving reference polypeptide best match to mass fingerprint (kDa)¹ ofreference polypeptide 88 49243545 55 81762012 38 82750440 37 49243435 3657286380 35 49245508 33 49243946 ¹Molecular weight as determined bySDS-PAGE.

TABLE 7 S. aureus SAAV1. NCBI sequence identifier of polypeptideidentified by the Molecular weight of computer algorithm as havingreference polypeptide best match to mass fingerprint (kDa) ¹ ofreference polypeptide 55 57286470 55 48874 37 49243435 33 49243946 ¹Molecular weight as determined by SDS-PAGE.

TABLE 8 S. aureus 2176. NCBI sequence identifier of polypeptideidentified by the Molecular weight of computer algorithm as havingreference polypeptide best match to mass fingerprint (kDa)¹ of referencepolypeptide 88 57285406 80 57285406 65 57285406 55 57286528 37 4948235836 57286380 35 15927153 33 57285658 32 57285658 ¹Molecular weight asdetermined by SDS-PAGE.

TABLE 9 S. aureus 1477. NCBI sequence identifier of polypeptideidentified by the Molecular weight of computer algorithm as havingreference polypeptide best match to mass fingerprint (kDa)¹ of referencepolypeptide 88 49482458 80 57285406 65 57285406 55 57286528 36 1592715335 49484031 ¹Molecular weight as determined by SDS-PAGE.

The polypeptides expressed by a reference microbe and referred to aboveby molecular weight can be obtained by growth of the reference microbeunder low metal conditions and the subsequent isolation of a polypeptideby the processes disclosed herein. A candidate polypeptide is isolatablefrom a microbe, preferably a gram positive microbe, more preferably, amember of the family Micrococcaceae, preferably, Staphylococcus spp.,more preferably, Staphylococcus aureus. Other gram positive microbesinclude Corynebacterium spp., Erysipelothrix spp., Mycobacterium spp.,and Erysipelothrix spp. A candidate polypeptide may also be producedusing recombinant, enzymatic, or chemical techniques.

Also provided by the present invention are whole cell preparations of amicrobe, where the microbe expresses one or more of the polypeptides ofthe present invention. The cells present in a whole cell preparation arepreferably, inactivated such that the cells cannot replicate, but theimmunogenic activity of the polypeptides of the present inventionexpressed by the microbe is maintained. Typically, the cells are killedby exposure to agents such as glutaraldehyde, formalin, or formaldehyde.

Compositions

A composition of the present invention may include at least onepolypeptide described herein, or a number of polypeptides that is aninteger greater than 1 (e.g., at least 2, at least 3, at least 4). Forexample, a composition can include 2, 3, 4, 5, or more isolated metalregulated polypeptides having molecular weights of 88 kDa, 55 kDa, 38kDa, 37 kDa, 36 kDa, 35 kDa, 33 kDa, or any subset or combinationthereof. A composition, can include polypeptides isolatable from 1microbe, or can be isolatable from a combination of 2 or more microbes.For instance, a composition can include polypeptides isolatable from 2or more Staphylocccocus spp., or from a Staphyloccacus spp. and adifferent microbe that is not a member of the genus Staphyloccocus. Thepresent invention also provides compositions including a whole cellpreparation, where the whole cell expresses one or more of thepolypeptides of the present invention. For instance, the whole cell canbe a Staphyloccocus spp. In some aspects, a composition can includewhole preparations from 2, 3, 4, 5, or 6 strains.

Optionally, a polypeptide of the present invention can be covalentlybound or conjugated to a carrier polypeptide to improve theimmunological properties of the polypeptide. Useful carrier polypeptidesare known in the art. The chemical coupling of polypeptides of thepresent invention can be carried out using known and routine methods.For instance, various homobifunctional and/or heterobifunctionalcross-linker reagents such as bis(sulfosoccinimidyl) suberate,bis(diazobenzidinine), dimethyl adipimidate, dimethyl pimelimidate,dimethyl superimidate, disuccinimidyl suberate, glutaraldehyde,m-maleimidobenzoyl-N-hydroxysuccinimide,sulfosuccinimidyl-4-(N-maleimidomethyl) cycloheane-1-carboxylate,sulfosuccinimidyl 4-(p-maleimido-phenyl) butyrate and(1-ethyl-3-(dimethyl-aminopropyl) carbodiimide can be used (see, forinstance, Harlow and Lane, Antibodies, A Laboratory Manual, generallyand Chapter 5, Cold Spring Harbor Laboratory, Cold Spring Harbor, NewYork, N.Y. (1988)).

The compositions of the present invention optionally further include apharmaceutically acceptable carrier. “Pharmaceutically acceptable”refers to a diluent, carrier, excipient, salt, etc, that is compatiblewith the other ingredients of the composition, and not deleterious tothe recipient thereof. Typically, the composition includes apharmaceutically acceptable carrier when the composition is used asdescribed herein. The compositions of the present invention may beformulated in pharmaceutical preparations in a variety of forms adaptedto the chosen route of administration, including routes suitable forstimulating an immune response to an antigen. Thus, a composition of thepresent invention can be administered via known routes including, forexample, oral; parental including intradermal, transcutaneous andsubcutaneous; intramuscular, intravenous, intraperitoneal, etc. andtopically, such as, intranasal, intrapulmonary, intramammary,intravaginal, intrauterine, intradermal, transcutaneous and rectally,etc. It is foreseen that a composition can be administered to a mucosalsurface, such as by administration to the nasal or respiratory mucosa(e.g. spray or aerosol), in order to stimulate mucosal immunity, such asproduction of secretory IgA antibodies, throughout the animal's body.

A composition of the present invention can also be administered via asustained or delayed release implant. Implants suitable for useaccording to the invention are know and include, for example, thosedisclosed in Emery and Straub (WO 01/37810 (2001)), and Emery et al.,(WO 96/01620 (1996)). Implants can be produced at sizes small enough tobe administered by aerosol or spray. Implants also include nanospheresand microspheres.

A composition of the present invention may be administered in an amountsufficient to treat certain conditions as described herein. The amountof polypeptides or whole cells present in a composition of the presentinvention can vary. For instance, the dosage of polypeptides can bebetween 0.01 micrograms (μg) and 300 mg, typically between 0.1 mg and 10mg. When the composition is a whole cell preparation, the cells can bepresent at a concentration of, for instance, 10² bacteria/ml, 10³bacteria/ml, 10⁴ bacteria/ml, 10⁵ bacteria/ml, 10⁶ bacteria/ml, 10⁷bacteria/ml, 10⁸ bacteria/ml, or 10⁹ bacteria/ml. For an injectablecomposition (e.g. subcutaneous, intramuscular, etc.) the polypeptidesmay be present in the composition in an amount such that the totalvolume of the composition administered is 0.5 ml to 5.0 ml, typically1.0-2.0 ml. When the composition is a whole cell preparation, the cellsare preferably present in the composition in an amount that the totalvolume of the composition administered is 0.5 ml to 5.0 ml, typically1.0-2.0 ml. The amount administered will vary depending on variousfactors including, but not limited to, the specific polypeptides chosen,the weight, physical condition and age of the animal, and the route ofadministration. Thus, the absolute weight of the polypeptide included ina given unit dosage form can vary widely, and depends upon factors suchas the species, age, weight and physical condition of the animal, aswell as the method of administration. Such factors can be determined byone of skill in the art. Other examples of dosages suitable for theinvention are disclosed in Emery et al., (U.S. Pat. No. 6,027,716).

The formulations may be conveniently presented in unit dosage form andmay be prepared by methods well known in the art of pharmacy. Methods ofpreparing a composition with a pharmaceutically acceptable carrierinclude the step of bringing the active compound (e.g., a polypeptide orwhole cell of the present invention) into association with a carrierthat constitutes one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing theactive compound into association with a liquid carrier, a finely dividedsolid carrier, or both, and then, if necessary, shaping the product intothe desired formulations.

A composition including a pharmaceutically acceptable carrier can alsoinclude an adjuvant. An “adjuvant” refers to an agent that can act in anonspecific manner to enhance an immune response to a particularantigen, thus potentially reducing the quantity of antigen necessary inany given immunizing composition, and/or the frequency of injectionnecessary in order to generate an adequate immune response to theantigen of interest. Adjuvants may include, for example, IL-1, IL-2,emulsifiers, muramyl dipeptides, dimethyl dioctadecyl ammonium bromide(DDA), avridine, aluminum hydroxide, oils, saponins, alpha-tocopherol,polysaccharides, emulsified paraffins (including, for instance, thoseavailable from under the tradename EMULSIGEN from MVP Laboratories,Ralston, Nebr.), ISA-70, RIBI and other substances known in the art. Itis expected that polypeptides of the present invention will haveimmunoregulatory activity and that such polypeptides may be used asadjuvants that directly act as T and/or B cell activators or act onspecific cell types that enhance the synthesis of various cytokines oractivate intracellular signaling pathways. Such polypeptides areexpected to augment the immune response to increase the protective indexof the existing composition.

In another embodiment, a composition of the invention including apharmaceutically acceptable carrier can include a biological responsemodifier, such as, for example, IL-2, IL-4 and/or IL-6, TNF, IFN-alpha,IFN-gamma, and other cytokines that effect immune cells. An immunizingcomposition can also include other components known in the art such asan antibiotic, a preservative an antioxidant, or a chelating agent.

Methods of Making

The present invention also provides methods for obtaining thepolypeptide described herein. The polypeptides and whole cells of thepresent invention are isolatable from a member of the familyMicrococcaceae, preferably, Staphylococcus spp., more preferably,Staphylococcus aureus. Other gram positive microbes from whichpolypeptides can be isolated include Corynebacterium spp.,Erysipelothrix spp., Mycobacterium spp., and Erysipelothrix spp.Microbes useful for obtaining polypeptides of the present invention andmaking whole cell preparations are commercially available from adepository such as American Type Culture Collection (ATCC). In addition,such microbes are readily obtainable by techniques routine and known tothe art. The microbes may be derived from an infected animal as a fieldisolate, and used to obtain polypeptides and/or whole cell preparationsof the present invention, or stored for future use, for example, in afrozen repository at −20° C. to −95° C., or −40° C. to −50° C., inbacteriological media containing 20% glycerol, and other like media.

When a polypeptide of the present invention is to be obtained from amicrobe, the microbe can be incubated under low metal conditions. Asused herein, the phrase “low metal conditions” refers to an environment,typically bacteriological media, which contains amounts of a free metalthat cause a microbe to express metal regulated polypeptides at adetectable level. As used herein, the phrase “high metal conditions”refers to an environment that contains amounts of a free metal thatcause a microbe to either not express one or more of the metal regulatedpolypeptides described herein at a detectable level, or to decreaseexpression of such a polypeptide. Metals are those present in theperiodic table under Groups 1 through 17 (IUPAC notation; also referredto as Groups I-A, II-A, III-B, IV-B, V-B, VI-B, VII-B, VIII, I-B, II-B,III-A, IV-A, V-A, VI-A, and VII-A, respectively, under CAS notation).Preferably, metals are those in Groups 2 through 12, more preferably,Groups 3-12. Even more preferably, the metal is iron, zinc, copper,magnesium, nickel, cobalt, manganese, molybdenum, or selenium, mostpreferably, iron.

Low metal conditions are generally the result of the addition of a metalchelating compound to a bacteriological medium, the use of abacteriological medium that contains low amounts of a metal, or thecombination thereof. High metal conditions are generally present when achelator is not present in the medium, a metal is added to the medium,or the computation thereof. Examples of metal chelators include naturaland synthetic compounds. Examples of natural compounds include plantphenolic compounds, such as flavenoids. Examples of flavenoids includethe copper chelators catechin and naringenin, and the iron chelatorsmyricetin and quercetin. Examples of synthetic copper chelators include,for instance, tetrathiomolybdate, and examples of synthetic zincchelators include, for instance, N,N,N′,N′-Tetrakis(2-pyridylmethyl)-ethylene diamine. Examples of synthetic iron chelatorsinclude 2,2′-dipyridyl (also referred to in the art as α,α′-bipyridyl),8-hydroxyquinoline, ethylenediamine-di-O-hydroxyphenylacetic acid(EDDHA), desferrioxamine methanesulphonate (desferol), transferrin,lactoferrin, ovotransferrin, biological siderophores, such as, thecatecholates and hydroxamates, and citrate. An example of a generaldivalent cation chelator is Chelex® resin. Preferably, 2,2′-dipyridyl isused for the chelation of iron. Typically, 2,2′-dipyridyl is added tothe media at a concentration of at least 300 micrograms/milliliter(μg/ml), at least 600 μg/ml, or at least 900 μg/ml. High levels of2,2′-dipyridyl can be 1200 μg/ml, 1500 μg/ml, or 1800 μg/ml.

The S. aureus genome encodes three Fur homologs: Fur, PerR, and Zur.While the Zur and PerR proteins appear to be primarily involved inregulating zinc homeostasis and peroxide stress genes, respectively, theFur protein has been demonstrated to regulate several iron-siderophoreuptake systems in response to iron limitation. The Fur protein alsoplays a role in oxidative stress resistance and virulence. It isexpected that a gram positive organism, preferably, an S. aureus, with amutation in a fur gene will result in the constitutive expression ofmany, if not all, of the metal regulated polypeptides of the presentinvention. The production of fur mutation in a gram positive,preferably, an S. aureus, can be produced using routine methodsincluding, for instance, transposon, chemical, or site-directedmutagenesis useful for generating gene knock-out mutations in grampositive bacteria.

The medium used to incubate the microbe and the volume of media used toincubate the microbe can vary. When a microbe is being evaluated for theability to produce one or more of the polypeptides described herein, themicrobe can be grows in a suitable volume, for instance, 10 millilitersto 1 liter of medium. When a microbe is being grown to obtainpolypeptides for use in, for instance, administration to animals, themicrobe may be grown in a fermentor to allow the isolation of largeramounts of polypeptides. Methods for growing microbes in a fermentor areroutine known to the art. The conditions used for growing a microbepreferably include a metal chelator, more preferably an iron chelator,for instance 2,2′-dipyridyl, a pH of between 6.5 and 7.5, preferablybetween 6.9 and 7.1, and a temperature of 37° C.

In some aspects of the invention, a microbe may be harvested aftergrowth. Harvesting includes concentrating the microbe into a smallervolume and suspending in a media different than the growth media.Methods for concentrating a microbe are routine and known in the art,and include, for example, filtration or centrifugation. Typically, theconcentrated microbe is suspended in an appropriate buffer. An exampleof a buffer that can be used contains Tris-base (7.3 grams/liter), at apH of 8.5. Optionally, the final buffer also minimizes proteolyticdegradation. This can be accomplished by having the final buffer at a pHof greater than 8.0, preferably, at least 8.5, and/or including one ormore proteinase inhibitors (e.g., phenylmethanesulfonyl fluoride).Optionally and preferably, the concentrated microbe is frozen at −20° C.or below until disrupted.

When the microbe is to be used as a whole cell preparation, theharvested cells may be processed using routine and known methods toinactivate the cells. Alternatively, when a microbe is to be used toprepare polypeptides of the present invention, the microbe may bedisrupted using chemical, physical, or mechanical methods routine andknown to the art, including, for example, boiling, french press,sonication, digestion of peptidoglycan (for instance, by digestion withlysozyme), or homogenization. An example of a suitable device useful forhomogenization is a model C500 B AVESTIN Homogenizer, (Avestin Inc,Ottawa Canada). As used herein, “disruption” refers to the breaking upof the cell. Disruption of a microbe can be measured by methods that areroutine and known to the art, including, for instance, changes inoptical density. Typically, a microbe is subjected to disruption untilthe percent transmittance is increased by 20% when a 1:100 dilution ismeasured. When physical or mechanical methods are used, the temperatureduring disruption is typically kept low, preferably at 4° C., to furtherminimize proteolytic degradation. When chemical methods are used thetemperature may be increased to optimize for the cell disruption. Acombination of chemical, physical, and mechanical methods may also beused to to solubilize the cell wall of microbe. As used herein, the term“solubilize” refers to dissolving cellular materials (e.g.,polypeptides, nucleic acids, carbohydrates) into the aqueous phase ofthe buffer in which the microbe was disrupted, and the formation ofaggregates of insoluble cellular materials. Without intending to belimited by theory, the conditions for solubilization are believed toresult in the aggregation of polypeptides of the present invention intoinsoluble aggregates that are large enough to allow easy isolation by,for instance, centrifugation.

The insoluble aggregates that include one or more of the polypeptides ofthe present invention may be isolated by methods that are routine andknown to the art. Preferably, the insoluble aggregates are isolated bycentrifugation. Typically, centrifugation of polypeptides, such asmembrane polypeptides, can be accomplished by centrifugal forces of100,000×g. The use of such centrifugal forces requires the use ofultracentrifuges, and scale-up to process large volumes of sample isoften difficult and not economical with these types of centrifuges. Themethods described herein provide for the production of insolubleaggregates large enough, to allow the use of continuous flowcentrifuges, for instance T-1 Sharples (Alfa Laval Separations,Warminster, Pa.), which can be used with a flow rate of 250 ml/minute as17 psi at a centrifugal force of 46,000×g to 60,000×g. Other large scalecentrifuges can be used, such as the tubular bowl, chamber, and discconfigurations. Such centrifuges are routinely used and known in theart, and are commercially available from such manufactures as Pennwalt,Westfalia and alpha-Laval.

The final harvested proteins are washed and/or dialyzed against anappropriate buffer using methods known in the art, for instancediafiltration, precipitation, hydrophobic chromatography, ion-exchangechromatography, or affinity chromatography, or ultra filtration andwashing the polypeptides, for instance, in alcohol, by diafiltration.After isolation, the polypeptides suspended in buffer and stored at lowtemperature, for instance, −20° C. or below.

In those aspects of the present invention where a whole cell preparationis to be made, after growth a microbe can be killed with the addition ofan agent such as glutaraldehyde, formalin, or formaldehyde, at aconcentration sufficient to inactivate the cells in the culture. Forinstance, formalin can be added at a concentration of 0.3% (vol:vol).After a period of time sufficient to inactivate the cells, the cells,can be harvested by, for instance, diafiltration and/or centrifugation,and washed.

Methods of Use

An aspect of the present invention is further directed to methods ofusing the compositions of the present invention. The methods includeadministering to an animal an effective amount of a composition of thepresent invention. The animal can be, for instance, avian (including,for instance, chickens or turkeys), bovine (including, for instance,cattle), caprine (including, for instance, goats), ovine (including, forinstance, sheep), porcine (including, for instance, swine), bison(including, for instance, buffalo), equine (including, for instance,horses), a companion animal (including, for instance, dogs or cats),members of the family Cervidae (including, for instance, deer, elk,moose, caribou and reindeer), or human.

In some aspects, the methods may further include additionaladministrations (e.g., one or more booster administrations) of thecomposition to the animal to enhance or stimulate a secondary immuneresponse. A booster can be administered at a time after the first,administration, for instance, 1 to 8 weeks, preferably 2 to 4 weeks,after the first administration of the composition. Subsequent boosterscan be administered one, two, three, four, or more times annually.Without intending to be limited by theory, it is expected that in someaspects of the present invention annual boosters will not be necessary,as so animal will be challenged in the field by exposure so microbesexpressing polypeptides present is the compositions having epitopes thatare identical to or structurally related to epitopes present onpolypeptides of the composition administered to the animal.

In one aspect, the invention is directed to methods for makingantibodies, for instance by inducing the production of antibody in ananimal, or by recombinant techniques. The antibody produced includesantibody that specifically binds at least one polypeptide present in thecomposition. In this aspect of the invention, an “effective amount” isan amount effective to result in the production of antibody in theanimal. Methods for determining whether an animal has producedantibodies that specifically bind polypeptides present in a compositionof the present invention can be determined as described herein. Thepresent invention further includes antibody that specifically bind to apolypeptide of the present invention, and compositions including suchantibodies. The method may be used to produce antibody that specificallybinds polypeptides expressed by a microbe other than the microbe fromwhich the polypeptides of the composition were isolated. As used herein,an antibody that can “specifically bind” a polypeptide is an antibodythat interacts with the epitope of the antigen that induced thesynthesis of the antibody, or interacts with a structurally relatedepitope. At least some of the polypeptides present in the compositionsof the present invention typically include epitopes that are conservedin the polypeptides of different species and different genera ofmicrobes. Accordingly, antibody produced using a composition derivedfrom one microbe is expected to bind to polypeptides expressed by othermicrobes and provide breed spectrum protection against gram positiveorganisms. Examples of gram positive microbes to which the antibody mayspecifically bind are Micrococcaceae, preferably, Staphylococcus spp.,more preferably, Staphylococcus aureus; members of the familyStreptococcaceae, preferably, Streptococcus pyogenes, Streptococcuspneumoniae, Streptotcoccus agalactiae, Streptococcus uberis,Streptococcus bovis, Streptococcus equi, or Streptococcus dysgalactiae;and Bacillus spp., Clostridium spp., Corynebacterium spp., Enterococcusspp., Erysipelothrix spp., Listeria spp., Microoccus spp., andMycobacterium spp., Kytococcus spp., and Erysipelothrix spp.

The present invention is also directed to the use of such antibody totarget a microbe expressing a polypeptide of the present invention or apolypeptide having an epitope structurally related to an epitope presenton a polypeptide of the present invention. A compound can be covalentlybound to an antibody, where the compound can be, for instance, a toxin.Likewise, such compounds can be covalently bound to a bacterialsiderophore to target the microbe. The chemical coupling or conjugationof an antibody of the present invention, or a portion thereof (such asan Fab fragment), can be carried out using known and routine methods. Inone aspect the invention is also directed to treating an infection in ananimal, including a human, caused by a gram positive microbe, preferablyby a member of the family Micrococcaceae, preferably, Staphyhcoccusspp., more preferably, S. aureus, members of the familyStreptococcaceae, preferably, Streptococcus pyogenes, Streptococcuspneumoniae, Streptococcus agalactiae, Streptococcus uberis,Streptococcus bovis, Streptococcus equi, or Streptococcus dysgalactiae,Bacillus spp., Clostridium spp., Corynebacterium spp., Enterococcusspp., Erysipelothrix spp., Kytococcus spp., Listeria spp., Micrococcusspp., Mycobacterium spp., and Erysipelothrix spp. As used herein, theterm “infection” refers to the presence of a gram positive microbe in ananimal's body, which may or may not be clinically apparent. An animalwith an infection by a member of the genus Staphylococcus that is notclinically apparent is often referred to as an asymptomatic carrier. Themethod includes administering an effective amount of the composition ofthe present invention to an animal having an infection caused by a grampositive microbe, and determining whether the number of microbes causingthe infection has decreased. Methods for determining whether aninfection is caused by a gram positive microbe are routine and known inthe art, as are methods for determining whether the infection hasdecreased.

In another aspect, the present invention is directed to methods fortreating one or more symptoms of certain conditions in an animal thatmay be caused by infection by a gram positive microbe, preferably by amember of the family Micrococcaceae, preferably, Staphylococcus spp.,more preferably, S. aureus; members of the family Streptococcacae,preferably, Streptococcus pyogenes, Streptococcus pneumoniae,Streptococcus agalactiae, Streptococcus uberis, Streptococcus bovis,Streptococcus equi, or Streptococcus dysgalactiae; Bacillus spp.,Clostridium spp., Corynebacterium spp., Enterococcus spp.,Erysipelothrix spp., Kytococcus spp., Listeria spp., Micrococcus spp.,Mycobacterium spp., and Erysipelothrix spp. The method includesadministering an effective amount of a composition of the presentinvention to an animal having or at risk of having a condition, orsymptoms of a condition, and determining whether at least one symptom ofthe condition is changed, preferably, reduced. Examples of conditionscaused by microbial infections include, for instance, mastitis,septicemia, pneumonia, meningoencephalitis, lymphangitis, dermatitis,genital tract infections, strangles, metritis, perinatal disease,pituitary abscesses, arthritis, bursitis, orchitis, cystitis andpyelonephritis, caseous lymphadenitis, tuberculosis, ulcerativelymphangitis, listeriosis, erysipelas, laminitis, anthrax, tyzzer'sdisease, tetanus, botulism, enteritis, malignant edema, braxy, bacillaryhemoglobinuria, enterotoxemia, necrotic skin lesions, and nosocomialinfections. Examples of conditions caused by S. aureus also include, forinstance, botryomycosis in horses, purulent synovitis and osteomyelitisin poultry, abortions in swine, and tick pyemia in lambs. Examples ofconditions caused by Streptococcus spp. also include, for instance, sorethroat, scarlet fever, impetigo, ulcerative endocarditis, rheumaticfever and post streptococcal glomerulonephritis cervicitis in humans,cervicitis in equine and swine, and meningitis and jowl abscesses inswine.

Treatment of symptoms associated with these conditions can beprophylactic or, alternatively, can be initiated after the developmentof a condition described herein. As used herein, the term “symptom”refers to objective evidence in a subject of a condition caused byinfection by a microbe. Symptoms associated with conditions referred toherein and the evaluations of such symptoms are routine and known to theart. Treatment that is prophylactic, for instance, initiated before asubject manifests symptoms of a condition caused by a microbe, isreferred to herein as treatment of a subject that is “at risk” ofdeveloping the condition. Typically, an animal “at risk” of developing acondition is an animal present in an area where animals having thecondition have been diagnosed and/or is likely to be exposed to amicrobe causing the condition. Accordingly, administration of acomposition can be performed before, during, or after the occurrence ofthe conditions described herein. Treatment initiated after thedevelopment of a condition may result in decreasing the severity of thesymptoms of one of the conditions, or completely removing the symptoms.In this aspect of the invention, an “effective amount” is an amounteffective to prevent the manifestation of symptoms of a disease,decrease the severity of the symptoms of a disease, and/or completelyremove the symptoms. The successful treatment of a gram positivemicrobial infection in an animal is disclosed in Example 5, whichdemonstrates the protection against disease caused by S. aureus in mousemodels by administering a composition of the present invention. Thesemouse models are a commonly accepted model for the study of humandisease caused by these microbes. The successful treatment of a grampositive microbial infection in an animal is also disclosed in Examples10-12, which demonstrates the protection against disease caused by S.aureus in cows by administering a composition of the present invention.

The present invention also provides methods for decreasing colonizationby gram positive microbes, for instance blocking the attachment sites ofgram positive microbe, including tissues of the skeletal system (forinstance, bones, cartilage, tendons and ligaments), muscular system,(for instance, skeletal and smooth muscles), circulatory system (forinstance, heart, blood vessels, capillaries and blood), nervous system(for instance, brain, spinal cord, and peripheral nerves), respiratorysystem (for instance, nose, trachea lungs, bronchi, bronchioceles,alveoli), digestive system (for instance, mouth, salivary glandsoesophagus liver stomach large and small intestine), excretory system(for instance, kidneys, ureters, bladder and urethra), endocrine system(for instance, hypothalamus, pituitary, thyroid, pancreas and adrenalglands), reproductive system (for instance, ovaries, oviduct, uterus,vagina, mammary glands, testes, and seminal vesicles), lymphatic/immunesystems (for instance, lymph, lymph nodes and vessels, mononuclear orwhite blood cells, such as macrophages, neutrophils, monocytes,eosinophils, basophils, lymphocytes t- and b-cells), and specific celllineages (for instance, precursor cells, epithelial cells, stem cells),and the like. Preferably, the gram positive microbe is a member of thefamily Micrococcaceae, preferably, Staphylococcus spp., more preferably,S. aureus; a member of the family Streptooccaceae, preferably,Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcusagalactiae, Streptococcus uberis, Streptococcus bovis, Streptococcusequi, or Streptococcus dysgalactiae; Bacillus spp., Clostridium spp.,Cornebacterium spp., Entercococus spp., Erysipelothrix spp., Kytococcusspp., Listeria spp., Micrococcus spp., Mycobacterium spp., andErysipelothrix spp. The method includes administering an effectiveamount of a composition of the present invention to an animal colonizedby, or at risk of being colonized by, a gram positive microbe. In thisaspect of the invention, an “effective amount” is an amount sufficientto decrease colonization of the animal by the microbe. Methods forevaluating the colonization of an animal by a microbe are routine andknown in the art. For instance, colonization of an animal's intestinaltract, by a microbe can be determined by measuring the presence of themicrobe in the animal's feces. It is expected that decreasing thecolonization of an animal by a microbe will reduce transmission of themicrobe to humans.

A composition of the invention can be used to provide for active orpassive immunization against bacterial infection. Generally, thecomposition can be administered to an animal to provide activeimmunization. However, the composition can also be used to induceproduction of immune products, such as antibodies, which can becollected from the producing animal and administered to another animalto provide passive immunity. Immune components, such as antibodies, canbe collected to prepare compositions (preferably containing antibody)from serum, plasma, blood, colostrum, etc. for passive immunizationtherapies. Antibody compositions including monoclonal antibodies and/oranti-idiotypes can also be prepared using known methods. Chimericantibodies include human-derived constant regions of both heavy andlight chains and murine-derived variable regions that areantigen-specific (Morrison et al., Proc. Natl. Acad. Sci. USA, 1984,81(21):6851-5; LoBuglio et al., Proc. Natl. Acad. Sci. USA, 1989,86(11):4220-4; Boolianne et al. Nature, 1984, 312(5995):643-6),Humanized antibodies substitute the murine constant and framework (FR)(of the variable region) with the human counterparts (Jones et al.,Nature, 1986, 321(6069):522-5; Riechmann et al., Nature, 1988,332(6162); 323-7; Verboeyen et al., Science, 1988, 239(4847); 1534-6;Queen et al., Proc. Natl. Acad. Sci. USA, 1989, 86(24): 10029-33;Daugerty et al., Nucleic Acids Res., 1991, 19(9): 2471-6.).Alternatively, certain mouse strains can be used that have beengenetically engineered to produce antibodies that are almost completelyof human origin; following immunization the B cells of these mice areharvested and immortalized for the production of human monoclonalantibodies (Bruggeman and Taussig, Curr. Opin. Biotechnol., 1997, 8(4);455-8; Lonberg and Huszar, Int. Rev. Immunol., 1995; 13(1):65-93;Lonberg et al., Nature, 1994, 368:856-9; Taylor et al., Nucleic AcidsRes., 1992, 20:6287-95). Passive antibody compositions and fragmentsthereof, e.g., seFv, Fab, F(ab′)₂ or Fv or other modified forms thereof,may be administered to a recipient in the form of serum, plasma, blood,colostrum, and the like. However, the antibodies may also be isolatedfrom serum plasma, blood, colostrum, and the like, using known methodsfor later use in a concentrated or reconstituted form such as, forinstance, lavage solutions, impregnated dressings and/or topical agentsand the like. Passive immunization preparations may be particularlyadvantageous for the treatment of acute systemic illness, or passiveimmunization of young animals that failed to receive adequate levels ofpassive immunity through maternal colostrum. Antibodies useful forpassive immunization may also be useful to conjugate to various drugs orantibiotics that could be directly targeted to bacteria expressingduring a systemic or localized infection a polypeptide of the presentinvention or a polypeptide having an epitope structurally related to artepitope present on a polypeptide of the present invention.

Animal models, in particular mouse models, are available forexperimentally evaluating the compositions of the present invention.These mouse models are commonly accepted models for the study of humandisease caused by members of the genus Staphylococcus, and S. aureus inparticular. In those cases where a members of the genus Staphylococcuscauses disease in an animal, for instance a cow, the natural host can beused to experimentally evaluate the compositions of the presentinvention.

Another aspect of the present invention provides methods for detectingantibody that specifically binds polypeptides of the present invention.These methods are useful in, for instance, detecting whether an animalhas antibody that specifically binds polypeptides of the presentinvention, and diagnosing whether an animal may have a condition causedby a microbe expressing polypeptides described herein, or expressingpolypeptides that share epitopes with the polypeptides described herein.Such diagnostic systems may be in kit form. The methods includecontacting an antibody with a preparation that include a polypeptide ofthe present invention to result in a mixture. The antibody may bepresent in a biological sample, for instance, blood, milk, or colostrum.The method leather includes incubating the mixture under conditions toallow the antibody to specifically bind the polypeptide to form apolypeptide:antibody complex. As used herein, the termpolypeptide:antibody complex refers to the complex that results when anantibody specifically binds to a polypeptide. The preparation thatincludes the polypeptides of the present invention may also includereagents, for instance a buffer, that provide conditions appropriate forthe formation of the polypeptide:antibody complex. Thepolypeptide-antibody complex is then detected. The detection ofantibodies is known in the art and can include, for instance,immunofluorescence or peroxidase. The methods for detecting the presenceof antibodies that specifically bind to polypeptides of the presentinvention can be used in various formats that have been used to detectantibody, including radioimmunoassay and enzyme-linked immunosorbentassay.

The present invention also provides a kit for detecting antibody thatspecifically binds polypeptides of the present invention. The antibodydetected may be obtained from as animal suspected to have an infectioncaused by a gram positive microbe, more preferably, a member of thefamily Micrococcaceae, preferably Staphylococcus spp., more preferably,S. aureus; Streptococcus spp., Bacillus spp., Clostridium spp.,Corynebacterium spp., Enterococcus spp., Erysipelothrix spp., Kytococcusspp., Listeria spp., Micrococcus spp., Mycobacterium spp., andErysipelothrix spp.

The kit includes at least one of the polypeptides of the presentinvention, or a numbers of polypeptides that is an integer greater than1 (e.g., at least 2, at least 3, etc.), in a suitable packaging materialin an amount sufficient for at least one assay. Optionally, otherreagents such as buffers and solutions needed to practice the inventionare also included. For instance, a kit may also include a reagent topermit defection of an antibody that specifically binds to a polypeptideof the present invention, such as a detectably labeled secondaryantibody designed to specifically bind to an antibody obtained from ananimal. Instructions for use of the packaged polypeptides are alsotypically included. As used herein, the phrase “packaging material”refers to one or more physical structures used to house the contents ofthe kit. The packaging material is constructed by well known methods,generally to provide a sterile, contaminant-free environment. Thepackaging material may have a label which indicates that thepolypeptides can be used for detecting antibody that specifically bindspolypeptides of the present invention. In addition, the packagingmaterial contains instructions indicating how the materials within thekit are employed to detect the antibody. As used herein, the term“package” refers to a container such as glass, plastic, paper, foil, andthe like, capable of holding within fixed limits the polypeptides, andother reagents, for instance a secondary antibody. Thus, for example, apackage can be a microtiter plate well to which microgram quantities ofpolypeptides have been affixed. A package can also contain a secondaryantibody, “Instructions for use” typically include a tangible expressiondescribing the reagent concentration or at least one assay methodparameter, such as the relative amounts of reagent and sample to beadmixed, maintenance time periods for reagent/sample admixtures,temperature, buffer conditions, and the like.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

EXAMPLES Example 1 Preparation of Iron Regulated Proteins LaboratoryScale

Compositions derived from different strains of Staphylococcus aureusincluding novel proteins expressed under iron-restriction and/or otherdegrees of metal ion chelation were evaluated for efficacy against avirulent challenge in mice. The efficacy of the composition wasevaluated by collecting data on the following parameters (1) theefficacy of each composition to provide homologous and heterologousprotection against a live virulent challenge in mice, (2) the efficacyof each composition to reduce necrotic skin lesions, and (3) theefficacy of compositions derived from Staphylococcus grown in repleteand deplete iron conditions to provide protection.

The Staphylococcus aureus strains evaluated in this study originatedfrom three animal species; avian, human and bovine. The avian isolateSAAV1 was a field isolate originating from a flock of diseased turkeyshaving a high degree of osteomyelitis and synovitis. The bovine isolates(strain 1477 and strain 2176) were isolated from two differentcommercial dairy herds having a high incidence of clinical mastitis. Thehuman isolate was obtained from the ATCC (strain 19636), and originatedfrom a patient having clinical osteomyelitis.

Master seed stocks of each isolate were prepared by inoculating theappropriate isolate into 200 ml of Tryptic Soy Broth (TSB, DifcoLaboratories, Detroit, Mich.) containing 300 μM 2,2-dipyribyl(Sigma-Aidrich St. Louis, Mo.). The culture was grown while stirring at200 rpm for 6 hours at 3° C., and collected by centrifugation at10,000×g. The bacterial pellet was re-suspended into 100 ml TSB brothcontaining 20% glycerol, and sterilely dispensed into 2 ml cryogenicvials (1 ml per vial) and stored at −90° C. until use.

Each master seed stock was expanded into a working seed. One vial ofeach master seed isolate was inoculated into 200 ml of Tryptic Soy Broth(TSB, Difco Laboratories, Detroit, Mich.) containing 1000 μM2,2-dipyridyl (Sigma-Aldrich St, Louis, Mo.). The culture was grownwhile stirring at 200 rpm for 6 hours at 37° C., and collected bycentrifugation at 10,000×g. The bacterial pellet was resuspended into100 ml TSB broth containing 20% glycerol, and sterilely dispensed into 2ml cryogenic vials (1 ml per vial) and stored −90° C. and use. Theworking seed was used for the production of compositions enriched withiron-regulated membrane proteins, including iron-regulated membraneproteins.

All strains were adapted to grow in highly iron-depleted media (i.e.,media containing very low levels of free iron). This was accomplished bysub-culturing the bacteria in TSB containing increasing concentrationsof 2,2-dipyridyl (from 300 to 1600 μM).

Proteins were prepared from bacteria as follows. The bacteria were grownfrom frozen working seed stocks by subculturing into 25 ml ofiron-deplete media (containing 1000 μM 2,2′-dyipyridyl) and iron-repletemedia, then incubated at 37° C. while shaking at 400 rpm. Following 12hours of incubation, 5 ml of each culture was transferred into 500 ml ofiron-deplete or iron-replete media pre-incubated at 37° C. Cultures wereincubated for 8 hours at 37° C. while shaking at 100 rpm, then cellswere pelleted by centrifugation at 10,000×g for 20 minutes. Bacterialpellets were resuspended in 100 ml of sterile physiological saline andcentrifuged at 10,000×g for 10 minutes. Pellets were then resuspended in45 ml of Tris-buffered saline, pH 7.2 (TBS; 25 mM Tris, 150 mM NaCl) andthe resulting bacterial suspensions were dispensed as 9-ml aliquots into5 individual tubes. One milliliter of TBS containing 50 units oflysostaphin (Sigma, St. Louis, Mo.) was added to each tube to give afinal volume of 5 units/ml. Following incubation at 37° C. for 30minutes while shaking at 200 rpm, 1 ml of TBS containing 0.1 mg oflysozyme (Sigma) was added to each tube. The bacterial suspensions werethen incubated for an additional 45 minutes while shaking at 200 rpm.Next, suspensions were centrifuged at 3050×g for 12 minutes at 4° C. topellet large cellular debris. The supernatants were collected byaspiration without disturbing the pellet. The supernatant was thencentrifuged at 39,000×g for 25 hours. The resulting pellets containingthe proteins were resupended into 200 μL. Tris buffer, pH 7.2, withoutsaline. The protein solution for each isolate were combined for a totalvolume of 1 ml and stored at −90° C.

The protein-enriched extracts derived from S. aureus weresize-fractionated on SDS-PAGE gels using a 4% stacking gel and 10%resolving gel. Samples for electrophoresis were prepared by combining 10μl of sample with 30 μl of SDS reducing sample buffer (62.5 mM Tris-HCLpH 6.8, 20% glycerol, 2% SDS, 5% β-mercaptoethanol) and boiled for 4minutes. Samples were electrophoresed at 18 mA constant current for 5hours at 4° C. using a Protein U xi cell power supply (BioRadLaboratories, Richmond, Calif., model 1000/500). The molecular weight ofeach individual protein as visually seen in the SDS-PAGE was estimatedusing a GS-800 densitometer (BioRad) using a broad range molecularweight marker as a reference standard (BioRad).

The SDS-PAGE patterns of the proteins from each isolate when grown inthe presence of 1000 μM dipyridyl showed a very different proteinexpression pattern compared to the same strain when grown in thepresence of 300 μM dipyridyl. For instance, when grown in 300 μMdipyridyl isolate SAAV1 resulted in metal regulated proteins of 90 kDa,84 kDa, 72 kDa, 66 kDa, 36 kDa, 32 kDa, and 22 kDa, while growth in 1600μM dipyridyl resulted in metal regulated proteins of 87.73 kDa, 54.53kDa, 38.42 kDa, 37.37 kDa, 35.70 kDa, 34.91 kDa, and 33.0 kDa. Likewise,when grown in 300 μM dipyridyl isolate 19636 resulted in proteins of 42kDa and 36 kDa, while growth in 1600 μM dipyridyl resulted in metalregulated proteins of 87.73 kDa, 54.53 kDa, 38.42 kDa, 37.37 kDa, 35.70kDa, 34.91 kDa, and 33.0 kDa. All conditions, including growth iniron-replete media, resulted in the expression of the following proteinsthat were presumably not metal regulated: 150 kDa, 132 kDa, 120 kDa, 75kDa, 58 kDa, 50 kDa, 44 kDa 43 kDa 41 kDa, and 40 kDa.

Furthermore, growth of the different strains of S. aureus in 1600 μMdipyridyl resulted in similar protein expression patterns. Thecompositions enriched in iron-regulated membrane proteins from the avianisolate (SAAV1) included proteins with molecular weights of 87.73 kDa,54.53 kDa, 38.42 kDa, 37.37 kDa, 35.70 kDa, 34.91 kDa, and 33.0 kDa. Themolecular weights of the proteins from the ATCC isolate 19636 wereessentially identical to those from the avian isolate. Both bovineisolates, when grown with 1600 μM 2,2-dipyridyl, expressed similarbanding profiles as the avian and ATCC isolates for the majority of theproteins (87.73 kDa, 54.53 kDa, 37.7 kDa, 35.70 kDa, 34.91 kDa, and 33.0kDa). However, neither of the bovine isolates produced the 38.42 kDaprotein seen with the avian and ATCC isolates, and the bovine isolatesexpressed three proteins (80.46 kDa, 65.08 kDa, and 31.83 kDa) notobserved with the avian and ATCC strains (see FIG. 1 and Table 10). Allconditions resulted in the expression of the following proteins thatwere not metal regulated: 150 kDa, 132 kDa, 120 KDa, 75 kDa, 58 kDa, 50kDa, 44 kDa, 43 kDa, 41 kDa, and 40 kDa.

TABLE 10 Molecular weights of metal regulated polypeptides obtained fromStaphylococcus aureus isolates. Avian Human Bovine Bovine SAAV1 196361477 2176 87.73 87.73 87.73 87.73 — — 80.46 80.46 — — 65.08 65.08 54.5354.53 54.53 54.53 38.42 38.42 — — 37.37 37.37 37.37 37.37 35.70 35.7035.70 35.70 34.91 34.91 34.91 34.91 33.0  33.0  33.0  33.0  31.83 31.83

Interestingly, there was no difference in the protein profiles asexamined by SDS-PAGE between the clarified supernatant and the bacterialpellet after treating the bacteria with lysostaphin/lysozyme. Both theextracted bacterial pellet and the supernatant had exactly the sameprotein profiles as examined by SDS-PAGE. This same observation was alsoseen when disrupting the bacterial cells using an AVESTIN homogenizer at30,000 psi. The resultant bacterial pellet, after slow speedcentrifugation was identical in its protein profile as compared to theclarified supernatant after high speed centrifugation at 30,000×g for2.0 hours at 4° C.

Example 2 Preparation of the Immunizing Compositions Derived fromStaphylococcus aureus

The proteins from the human isolate ATCC 19636 and the bovine isolate1477, grown in iron-deplete conditions and prepared as described inExample 1, were used to formulate two vaccine compositions. The proteinsfrom the ATCC isolate had molecular weights of 87.73 kDa, 54.53 kDa,38.42 kDa, 37.37 kDa, 35.70 kDa, 34.91 kDa, and 33.0 kDa, while thebovine isolate expressed proteins having molecular weights 87.73 kDa,80.46 kDa, 65.08 kDa, 54.53 kDa, 37.37 kDa, 35.70 kDa, 34.91 kDa, 33.0kDa, and 31.83. Each composition also contained the following proteinsthat were not metal regulated: 150 kDa, 132 kDa, 120 kDa, 75 kDa, 58kDa, 50 kDa, 44 kDa, 43 kDa, 41 kDa, and 40 kDa. Stock vaccines wereprepared from the two strains by emulsifying each aqueous proteinsuspension (500 μg total protein/ml) into a commercial adjuvant(EMULSIGEN, MVP Laboratories, Ralston, Nebr.) using an IKA Ultra TurraxT-50 homogenizing vessel (IKA, Cincinnati, Ohio) to give a final dose of50 μg total protein in a 0.1 ml injectable volume with an adjuvantconcentration of 22.5% vol/vol. As a control vaccination, a proteincomposition was prepared from the bovine isolate 1477 grown underiron-replete conditions (TSB supplemented with 300 μM ferric chloride)as described is Example 1. A placebo vaccine was prepared bysubstituting physiological saline for the aqueous protein suspension inthe above protocol.

Example 3 Moose Vaccination

Seventy (N=70) female CF-1 mice obtained from Harlan BreedingLaboratories (Indianapolis, Ind.) weighing 16-22 grams were equallydistributed into 7 groups (10 mice/group). Mice were housed inpolycarbonate mouse cages (Ancore Corporation, Bellmore, N.Y.). A singlecage was used for each treatment group and food and water was suppliedad libitum to all mice. All mice were vaccinated intraperitoneally with0.1 ml of the appropriate composition two times at 14 day intervals asfollows:

Group-1: Placebo-Vaccinated

Group-2: Vaccinated with ATCC 19636 proteins expressed underiron-restriction.

Group-3: Placebo-Vaccinated

Group-4: Vaccinated with Bovine 1477 proteins expressed underiron-restriction.

Group-5: Vaccinated with Bovine 1477 proteins expressed underiron-restriction.

Group-6: Vaccinated with ATCC 19636 proteins expressed underiron-restriction.

Group-7: Bovine 1477 FeCl₃-Vaccinated, where “Bovine 1477 FeCl₃” refersto proteins obtained from Bovine 1477 grown in TSB supplemented with 300μM ferric chloride.

Example 4 Preparation of Challenge Organists

The previously described Staphylococcus aureus strains ATCC 19636 andstrain 1477 were used as challenge organisms. Briefly, the isolates fromfrozen stocks (previously described) were streaked onto blood agarplates and incubated at 37° C. for 18 hours. A single colony of eachisolate was subcultured into 50 ml Tryptic Soy Broth (Difco) containing1600 μM 2,2′ dipyridyl. The cultures were incubated at 3° C. for 6 hourswhile rotating at 200 rpm, then centrifuged at 10,000×g for 10 minutesat 4° C. to pellet the bacteria. The final pellets were washed twice bycentrifugation in TBS at 4° C. The final pellets were resuspended in TBSto an optical density of 42% Transmittance (T) at 562 nm in a volume ofapproximately 25 ml of TBS and used for challenge. Just prior tochallenge, 1 ml of these bacterial suspensions was serially diluted andplated on agar to enumerate the number of colony-forming units (CFU) permouse dose.

Example 5 Challenge

Fourteen days after the second vaccination, mice in all groups (1-7)were subcutaneously challenged in the back of the neck with 0.1 ml ofthe appropriate organism. The seven groups of mice were challenged asfollows:

Group-1 (Placabo-Vacinated): Challenged with ATCC 19636

Group-2 (Vaccinated with ATCC 19636 proteins expressed underiron-restriction): Challenged with ATCC 19636

Group-3 (Placebo-Vaccinated): Challenged with Bovine 1477

Group-4 (Vaccinated Bovine 1477 proteins expressed underiron-restriction): Challenged with Bovine 1477

Group-5 (Vaccinated Bovine 1477 proteins expressed underiron-restriction): Challenged with ATCC 19636

Group-6 (Vaccinated ATCC 19636 proteins expressed underiron-restriction); Challenged with Bovine 1477

Group-7 (Bovine 1477 FeCl₃-Vaccinated): Challenged with Bovine 1477

As determined by the enumeration protocol described in Example 4, theconcentration of S. aureus 19636 used for challenge was 1.35×10⁸ CFU permouse dose, and the concentration of S. aureus 1477 used for challengewas 1.65×10⁸ colony CFU per mouse dose. Morbidity, mortality and grosspathology were recorded daily for 7 days after challenge.

When comparing the mice challenged with the ATCC 19636 isolate, 70% ofthe placebo-vaccinated Group 1 mice died within 7 days of challenge(Table 11 and FIG. 2). This demonstrated that strain 19636 caused a highrate of mortality in mice at the dose level administered. In contrast tothe mice in Group 1, only 10% of the mice in Group 2 died within 7 dayspost-challenge. These results illustrated that the mice challenged withstrain 19636 were significantly protected by vaccination with the 19636composition (p=0.020, Fischer's Exact test). Furthermore, a Kaplan-Meieranalysis of the time-to-death data indicated that the vaccine affordedsignificant (p=0.0042, logrank test) protection against homologouschallenge (FIG. 3). In addition, only 20% of the mice in Group 5 diedwithin 7 days of challenge, indicating that the bovine 1477 compositionoffered significant protection against challenge with the ATCC 19636strain (p=0.015 logrank test for mortality). When the data was analyzedby a Kaplan-Meier survival curve and logrank test (FIG. 4), theprotection against mortality was determined to be significant (p=0.015logrank test for mortality), indicating that the vaccine compositionderived from strain 1477 provided heterologous-protection againstchallenge with strain 19636.

TABLE 11 Mortality of Vaccinated and Non-Vaccinated Mice FollowingChallenge with Staphylococcus aureus (human ATCC isolate 19636 andbovine isolate 1477). Percent Groups # Mice # Dead mortality (%)Group-1* (Placebo, ATCC 19636 10 7/10 70 Chlg) Group-2* (ATCC 19636, 101/10 10 Homologous Chlg) Group-3* (Placebo, Bovine 1477 10 2/10 20 Chlg)Group-4* (Bovine 1477, 10 1/10 10 Homologous Chlg) Group-5* (Bovine1477, 10 2/10 20 Heterologous Chlg) Group-6* (ATCC 19636, 10 0/10 0Heterologous Chlg) Group-7* (Bovine 1477 FeCl₃, 10 2/10 20 Bovine 1477Chlg) *Group-1, (Placebo-Vaccinated/Challenged with ATCC 19636) *Group-2(Vaccinated with ATCC 19636 proteins expressed underiron-restriction/Challenged with ATCC 19636) *Group-3(Placebo-Vaccinated/Challenged with Bovine 1477) *Group-4 (Vaccinatedwith Bovine 1477 proteins expressed under iron-restriction/Challengedwith Bovine 1477) *Group-5 (Vaccinated with Bovine 1477 proteinsexpressed under iron-restriction/Challenged with ATCC 19636) *Group-6(Vaccinated with ATCC 19636 proteins expressed underiron-restriction/Challenged with Bovine 1477) *Group-7 (Bovine 1477FeCl₃ -Vaccinated/Challenged with Bovine 1477)

When comparing the mice challenged with the bovine 1477 isolate, only20% of the mice in the placebo-vaccinated group (Group 3) died within 7days of challenge. However, challenge with the bovine 1477 isolateelicited the development of necrotic skin lesions on 6 (75%) of thesurviving mice of Group 3. These lesions were measured and the averagesize of the lesions on the surviving mice was 18.5 mm (Table 12). Incontrast, 20% of the Group 4 mice died within 7 days of challenge, butonly three (38%) of the surviving mice developed lesions (averagediameter, 2.7 mm). These results indicate that the bovine 1477composition offered significant homologous protection againstdevelopment of lesions in the mice challenged with the bovine strain1477 (p=0.009, Student's t-test). In addition, vaccination, with theATCC 19636 composition protected against challenge with strain 1477,since no mice died in Group 6 and only three (30%) of the mice developedskin lesions (average diameter, 3.7 mm). Taken together, the reducedmortality and/or lesion development in the mice in Groups 5 and 6demonstrate the significant cross-protective nature of the compositionsderived from strains 19636 and 1477 (p=0.012, Students t-test based onlesion size). In demonstration of the efficacy of the composition ascompared to the non-iron regulated proteins, 20% of the mice in Group 7died and 4 of the survivors developed skin lesions (average diameter,15.8 mm). The mice of Group 7 demonstrated some degree of protection byvaccination with the proteins of the 1477 isolate since fewer micedeveloped lesions compared to the placebo-vaccinated Group 3. However,the skin lesions observed on the mice in group 7 were more frequent andof a larger diameter than the lesions an the mice of Group 4, indicatingthat, relative to proteins isolated from cells grown under iron-repleteconditions, the proteins isolated from bacteria grown under ironrestriction offered superior protection against so identical challenge.

TABLE 12 The Induction of Necrotic Lesions in Mice Seven DaysPost-Challenge with Staphylococcus aureus (ATCC Isolate 19636 and/orBovine Isolate 1477) Group-1 Group-2 Group-3 Group-4 Group-5 Group-6Group-7 Lesion diameter (millimeter) per mouse No No lesion 26 5 5 5 25lesion No No lesion 25 2 No lesion 5 25 lesion No No lesion 24 1 Nolesion 1 10 lesion Dead No lesion 24 No lesion No lesion No 3 lesionDead No lesion 7 No lesion No lesion No No lesion lesion Dead No lesion5 No lesion No lesion No No lesion lesion Dead No lesion No No lesion Nolesion No No lesion lesion lesion Dead No lesion No No lesion No lesionNo No lesion lesion lesion Dead No lesion Dead No lesion Dead No Deadlesion Dead Dead Dead Dead Dead No Dead lesion Average lesion diameter(mm) among surviving mice 0 0 18.5 2.7 5 3.7 15.8 Group-1,(Placebo-Vaccinated/Challenged ATCC 19636) Group-2 (Vaccinated with ATCC19636 proteins expressed under iron-restriction/Challenged ATCC 19636)Group-3 (Placebo-Vaccinated/Challenged Bovine 1477) Group-4 (Vaccinatedwith Bovine 1477 proteins expressed under iron-restriction/ChallengedBovine 1477) Group-5 (Vaccinated with Bovine 1477 proteins expressedunder iron-restriction/Challenged ATCC 19636) Group-6 (Vaccinated withATCC 19636 proteins expressed under iron-restriction/Challenged Bovine1477) Group-7 (Bovine 1477 FeCl₃ Vaccinated/Challenged Bovine 1477)

The cross-protective nature of the proteins observed in the mousechallenge study is supported by the similar molecular weights of theproteins from the S. aureus strains described in Example 1 (FIG. 1).Although there were noticeable differences in the SDS-PAGE profile ofthe proteins from the bovine-derived isolates, specifically the absenceof a 38.4 kDa protein and the presence of 3 additional proteins, theproteins from both strains 1477 and ATCC 19636 elicited heterologousprotection. These results indicate that the similar proteins betweenstrains 19636 and 1477 are likely responsible for the cross-protectionobserved in Groups 5 and 6. By contrast, the protein profiles fromstrain 1477 grown under iron-deplete and iron-replete conditions areobservably different. Those proteins isolated under iron-depletedconditions are more protective when compared to the proteins isolatedunder iron-replete conditions, demonstrated by the seduction in lesiondevelopment among the once of Croup 4 compared to the mice of Group 7.

Example 6

In mammals, it has been shown that the response to tissue injury orbacterial infection results in as acute inflammatory response. Thisresponse increases capillary permeability and phagocytic infiltrationresulting in the clinical signs recognized as inflammation; swelling,fever, pain and redness; if left uncontrolled, this may lead to death.The activation of humoral factors and the release of cytokines mediatesystemic events collectively known as the acute phase protein responsewhich results in a cascade of physiological and biochemical events. Theduration of this response is directly related to the severity of theinjury and magnitude of the systemic infection. It has beenwell-documented that during bacterial sepsis, major surgery, burns andother bodily trauma there is an alteration in the concentration of anumber of metal ions in serum such as, iron, copper, and zinc. Forinstance, dating the acute phase of an infection there is a decrease inplasma levels of iron and zinc and an increase in copper. The alterationof these trace metal ions in serum may directly affect the severity orprogression of any bacterial infection.

In this study we examined the expression of proteins of Staphylococcusaureus under various conditions of metal ion restriction in order tomimic the expression of novel proteins that may be expressed duringsystemic invasion. The Staphylococcus aureus strains evaluated in thisstudy originated from clinical samples of three different species ofanimal; avian (strain SAAV1), human (strain 19636), and bovine (strains1477 and 2176). Briefly, cultures of each isolate were prepared frommaster seed stocks in 200 ml of Tryptic Soy Broth (TSB). Each culturewas grown while stirring at 200 rpm for 6 hours at 37° C. Ten ml of eachculture were transferred into 500 ml of deplete TSB containing one offour metal ion chelators; 2,2-dipyridyl (Dp), 2-pyridylmethyl-ethylenediamine (TPEN), catechin, and naringenin (all obtained from Sigma, St.Louis, Mo.). In addition each culture was also grown in cation-repletemedia containing ferric chloride, zinc chloride and/or copper chlorideprepared 300 μM concentrations. The metal ion chelators were used at thefollowing concentration; 2,2-dipyridyl (800 μM), catechin and naringeninwere used at 300 μM, and 2-pyridylmethyl-ethylene diamine was used at aconcentration of 100 μM. Cultures were grown with each chelator for 8hours, at which point the culture was subcultured a second time for anadditional 12 hours. Each culture was subcultured for three consecutivepasses at 12-hour intervals. At the end of the third pass, each culturewas harvested by centrifugation at 10,000×g for 20 minutes. Each culturewas washed twice by centrifugation at 10,000×g and resuspended in 20 mlTris-buffered saline, pH 7.2 at 4° C.

Each bacterial pellet was resuspended in 45 ml of Tris-buffered saline,pH 7.2 (25 mM Tris and 150 mM NaCl) and the resulting bacterialsuspensions were dispensed as 9-ml aliquots into 5 individual tubes,twenty tubes total. One milliliter of TBS containing 50 units oflysostaphin (Sigma, St. Louis, Mo.) was added to each tube to give afinal concentration of 5 units/ml. Following incubation at 37° C. for 30minutes while shaking at 200 rpm, 1 ml of IBS containing 0.1 mg oflysosyme (Sigma) was added to each tube. The bacterial suspensions werethen incubated for an additional 45 minutes while shaking at 200 rpm.Next, suspensions were centrifuged at 3050×g for 12 minutes at 4° C. topellet large cellular debris. The supernatants were collected byaspiration without disturbing the pellet. The supernatant was thencentrifuged at 39,000×g for 2.5 hours. The resulting pellets, enrichedfor petal-regulated membrane proteins, were resuspended in 200 μL Trisbuffer, pH 7.2. The protein solutions for each isolate were combined fora total volume of 1 ml and stored at −90° C.

The proteins obtained from the SAAV1, 19636, 1477 and 2176 S. aureusisolates grown under iron, zinc and copper deplete conditions includedmetal-regulated polypeptides.

Cell extracts, derived from each isolate were size-fractionated onSDS-PAGE gels using a 4% stacking gel and 10 μl resolving gel. Samplesfor electrophoresis were prepared by combining 10 μl of sample with 30μl of SDS reducing sample buffer (62.5 mM Tris-HCL ph 6.8, 20% glycerol,2% SDS, 5% beta-mercaptoethanol) boiled for 4 minutes. Samples wereelectrophoresed 18 mA of constant current for 5 hours at 4° C. using aProtein II xi cell power supply (BioRad Laboratories, Richmond, Calif.,model 1000/500).

The SDS-PAGE patterns of the proteins grown under zinc and/or copperchelation showed unique handing patterns in all isolates that weredifferent when compared to the same isolates grown underiron-restriction in the presence of 2,2′-dyipyridyl. For example, whenthe 19636 isolate was grown under iron-restriction or in the presence ofthe chelator 2,2′-dyipyridyl, unique iron-regulated proteins wereexpressed at the 87.73 kDa, 54.53 kDa, 38.42 kDa, 37.37 kDa, 35.70 kDa,34.91 kDa and 33.0 kDa regions. These proteins were downregulated whenthe isolate was grown in the presence of ferric chloride. However, whenthe same isolate was grown in the presence of the zinc and or copperchelator, a novel subsets of proteins was expressed relative to theproteins expressed under iron-restriction; the new proteins havingmolecular weights of approximately 115 kDa, 88 kDa, 80 kDa, 71 kDa, 69kDa, 35 kDa, 30 kDa, 29 kDa and 27 kDa. In addition, an 87.73 kDaprotein was expressed under conditions of iron restriction orcopper-restriction but not when cultures were zinc-restricted. Theproteins expressed under iron-restriction appeared to be downregulatedwhen growth was under either zinc-restriction and/or copper-restrictionbut not completely shut off as seen when the isolate was grown in ferricchloride.

It appears that there are novel proteins expressed when the organism isgrown under copper-restriction and/or zinc-restriction that are notexpressed when the same isolate is grown under iron-restrictedconditions. Since transitional metals are used by organisms to buildenzymes that catalyze various biochemical reactions, the metal ions mayplay a vital role in microbial survival during a systemic infection. Itis perhaps for this reason that during sepsis there is a transientdecrease in the availability of these transitional metals, making themunavailable for growth of the organism. These novel proteins could verywell enhance the protective efficacy of the existing composition grownunder non-restriction because they may also be expressed by the bacteriaunder the metal ion restriction experienced during systemic invasion.

Example 7 Compositions of the Present Invention can also be ProducedUnder Large Scale Commercial Conditions

Fermentation

A cryogenic vial of the working seed (2 ml at 10⁹ CFU/ml) as describedin Example 1 was used to inoculate 500 ml of Tryptic Soy Broth (TSB)without dextrose (Difco) pre-warmed to 37° C. containing 0.125 g/l2,2-dipyridyl (Sigma), 2.7 grams BiTek yeast extract (Difco) andglycerol (3% vol/vol). The culture was incubated at 37° C. for 12 hourswhile stirring at 200 rpm at which time it was used to inoculate 2liters of the above media and allowed to grow for an additional 4 hoursat 37° C. This culture was used to inoculate a 20-liter VIRTIS bench-topfermentor, (Virtis, Gardiner, N.Y.) charged with 13 liters of theabove-described media. The pH was held constant between 6.9 and 7.1 byautomatic titration with 50% NaOH and 10% HCL. The stirring speed wasadjusted at 400 rev/minute, and the culture aerated with 11 litersair/minute at 37° C. Foaming was controlled automatically by theaddition of 11 ml defoamer (Mazu DF 204 Chem/Serv, Minneapolis, Minn.).The culture was allowed to grow continuously at these conditions for 4hours at which time was sterilely pumped into a 150-liter fermentor (W.B. Moore, Easton, Pa.). The fermentor was charged with 120 literstryptic soy broth without dextrose (3,600.0 grams), BiTek yeast extract(600 grams), glycerol (3,600 ml), 2,2-dypyrdyl (3.0 grams) and Mazu DF204 defoamer (60 ml). The parameters of the fermentation were asfollows: dissolved oxygen (DO) was maintained at 30%+/−10% by increasingagitation to 220 rev/minute sparged with 60 liters of air/minute and 10pounds per square inch (psi) back pressure. The pH was held constantbetween 6.9 and 7.1 by automatic titration with 50% NaOH and 10% HCL andthe temperature maintained at 37° C. At hour 4.5 (OD₅₄₀ 8-9) of thefermentation the culture was transferred to a 1,500 liter New BrunswickScientific fermentor IF-15000 charged with 1200 liters tryptic soy brothwithout dextrose (36,000 grams), BiTek yeast extract (6,000 grams),glycerol (36,000 ml), 2,2-dypyrdyl (30.0 grams) and Mazu DF 204 defoamer(600 ml). The parameters of the fermentation were as follows: dissolvedoxygen (DO) was maintained at 60%+/−10% with supplemental oxygen byincreasing agitation to 300 rev/minute sparged with 300 to 1100 litersof air/minute and 5 pounds per square inch (psi) back pressure. Asfermentation progressed supplemental oxygen was added from 0-90liters/minute to assist in the control of dissolved oxygen. The pH washeld constant between 6.9 and 7.4 by automatic titration with 50% NaOHand 10% HCL and the temperature was maintained at 37° C.

At approximately 5 hours post inoculation of the large fermentor theculture was supplemented with additional nutrients by feeding 70 litersof media containing 18,000 grams TSB without dextrose, 3,000 grams yeastextract 30.0 grams 2,2-dipyridyl and 18,000 ml of glycerol. The rate offeed was adjusted to approximately 28 liters/hour while increasingagitation. At the end of the feed the fermentation was allowed tocontinue for an additional 4 hours at which point the fermentation wasterminated by lowing the temperature of the fermentor to 18° C. (OD₅₄₀35-40 at a 1:100 dilution).

Harvest

The bacterial fermentation was concentrated and washed using a PallFiltron Tangential Flow Maxisel-25 (Pall Filtron Corporation, Nortboro,Mass.) equipped with three 30 ft² Alpha 300-K open channel filters,catalog No. AS300C5, (Pall Filtron) connected to a Waukesha Model U-60feed pump (Waukesha Cherry-Burrell, Delevan, Wis.) The original culturevolume of 1250 liters was reduced to 50 liters (2.5 liters/minute) usinga filter inlet pressure of 30 psi and a retentate pressure of 5-6 psi.The bacterial retentate was adjusted back up to 150 liters usingTris-buffered Saline pH 8.5 and then concentrated again to 50 liters tohelp remove any contaminating exogenous proteins, such as exoproteins toinclude secreted toxins and proteases. The elevated pH of thetris-buffered saline helps prevent much of the proteolytic degradationthat can occur during storage of the whole cell suspension. Proteaseinhibitors may be used instead of, or in addition to, an elevated pH.The retentate was mixed thoroughly while in the 200-liter tank using abottom mount magnetically driven mixer. The retentate was sterilelydispensed (3.5 liters) into sterile 4 liter Nalgene containers No. 2122and placed into a −20° C. freezer for storage as a breaking point in themanufacture, or could be further processed. The pellet mass wascalculated by centrifuging 30 ml samples of the fermented culture andfinal harvest. Briefly, pre-weighted 50 ml Nalgene conical tubes werecentrifuged at 39,000×g for 90 minutes in a Beckman J2-21 centrifugeusing a JA-21 rotor (Beckman Instruments, Palo Alto Calif.). At the endof the run, the supernate was poured off and the tubes were weighedagain. The pellet mass was calculated for each stage. The fermentationprocess yielded a wet pellet mass of approximately 60 kilograms.

Disruption

Eighty kilograms of bacterial cell slurry in Tris-buffered Saline pH 8.5was aseptically transferred into a steam in place 1000 liter jacketedprocess tank (Lee, Model 259LU) with a top mounted mixer (Eastern, ModelTME-1/2, EMI Incorporated, Clinton, Conn.) containing 900 liters TBS pH8.5. The bulk bacterial suspension was chilled to 4° C. with continuousmixing for 18 hours at 200 rpm at which time was disrupted byhomogenization. Briefly, the 1000 liter tank containing the bacterialsuspension was connected to a model C-500 B AVESTIN Homogenizer,(Avestin Inc, Ottawa Canada). A second 1000 liter jacketed process tank(empty) was connected to the homogenizer such that the fluid in theprocess tank could be passed through the homogenizer, into the emptytank and back again, allowing for multiple homogenizing passes whilestill maintaining a closed system. The temperature during homogenizationwas kept at 4° C. At the start of the first pass, fluid was circulatedat 70 psi via a Waukesha model 10DO pump (Waukesha) through thehomogenizer (500 gallons/hour), while the homogenizer pressure wasadjusted to 30,000 psi. Prior to the first pass, two pre-homogenizingsamples were withdrawn from the homogenizer to establish a baseline fordetermining the degree of disruption and monitoring of pH. The degree ofdisruption was monitored by transmittance (% T at 540 nm at 1:100dilution) compared to the non-homogenized sample. The number of passesthrough the homogenizer was standardized to give a final percenttransmittance between 78-91% T at a 1:100 dilution preferably between86-91%. After homogenization, the tank was removed from the homogenizerand put onto a chiller loop at 4° C. and mixed at 240 rpm.

Protein Harvest

The disrupted bacterial suspension containing the iron-regulatedproteins as illustrated in FIG. 1 were collected by centrifugation usingT-1 Sharples, (Alfa Laval Seperations, Warminster, Pa.). Briefly, the1000 liter jacketed process tank containing the disrupted bacterialhomogenate was fed into 12 Sharples with a feed rate of 250 ml/minute at17 psi at a centrifugal force of 60,000×g. The effluent was collectedinto a second 1000 liter jacketed process tank through a closed sterileloop allowing for multiple passes through the centrifuges whilemaintaining a closed system. The temperature during centrifugation waskept at 4° C. The homogentae was passed 8 times across the centrifuges.Approximately 50% of the protein was collected after the second pass, atwhich point, the homogenate fluid was concentrated in ⅓ of its originalvolume, which shortened the process time for the next 6 passes. Thehomogenate tank was aseptically disconnected from the centrifuges andconnected to a Millipore Pellicon Tangential Flow Filter assembly(Millipore Corporation, Bedford, Mass.), equipped with a 25 ft²screen-channel series Alpha 30K Centrasette filter (Pall Filtron)connected to a Waukesha Model U30 feed pump for concentration. Afterconcentration, centrifugation was continued until the process wascompleted. Protein was collected after each pass. The protein wascollected, resuspended and dispensed in 50 liters Tris-buffered salinepH 8.5 containing 0.15% formulin (Sigma) as preservative.

Diafiltration

The protein suspension was washed by diafiltration at 4° C. to removeany exogenous proteins (proteases, toxins, cytoplasmic and metabolicenzymes etc). Briefly, the 50 liters of protein was sterilelytransferred into a 200 liter process tank containing 150 liters sterileTris-buffer saline, pH 8.5 equipped with a bottom mount Dayton mixer.Model 2Z846 (Dayton Electric, Chicago, Ill.) rotating at 125 rev/minute.The process tank was sterilely connected to a Millipore PelliconTangential Flow Filter assembly (Millipore Corporation), equipped with a25 ft² screen-channel series Alpha 30K Centrasette filter (Pall Filtron)connected to a Waukesha Model U30 feed pump. The 200 liter proteinsolution was concentrated by filtration to a target volume 50 liters atwhich point 150 liters of sterile saline was added. The proteinsuspension was then concentrated to approximately 50 liters. The proteinconcentrate was stored in a 50 liter jacketed process tank equipped witha top mounted mixer and stored at 4° C.

It is interesting to note that the composition derived from the largescale process using homogenization as a means of disruption generatedidentical banding profiles as examined by SDS-PAGE as compared to thesmaller scale process described in Example 1. These results show thatlysostaphin could be replaced as the bacterial lysis agent using theAVESTIN homogenizer C500-B. This discovery allows for the low costgeneration of large volumes of iron-regulated proteins fromstaphlylococci.

Example 8 Hyper-Immunization of Mice and Preparation of PolyclonalAntibody

Passive immunization with purified antibody isolated from micevaccinated with proteins derived from S. aureus strains 19636 grownunder iron-limiting conditions was protective against a homologous andheterologous S. aureus challenge. Fifteen adult CD1 mice were vaccinatedas described in Example 3 wish the protein composition derived from S.aureus strain ATCC 19636 grown under iron-deplete conditions asdescribed in Examples 1 and 2. Mice were vaccinated intraperitoneally 3times at 7 day intervals with 50 μg of protein composition at eachvaccination. Seven days after the third immunization, mice were bledcompletely by cardiac puncture. Serum was pooled and antibody purifiedusing standard ammonium sulfate precipitation. Exogenous serum proteinswere removed first prior to antibody precipitation by adding 0.5 volumesof saturated ammonium sulfate pH 7.2. The solution was stirred at 100rpm for 24 hours at 4° C. The solution was again centrifuged at 3000×gfor 30 minutes. The supernatant was collected and precipitated again byadding enough saturated ammonium sulfate to bring the finalconcentration to 55% saturation. The solution was stirred at 100 rpm for24 hours at 4° C. The precipitate was centrifuged at 3000×g for 30minutes. The final pellet from each sample was resuspended into 2 ml PBSpH 7.2. The precipitated antibodies were then dialyzed using a 50,000molecular cut off dialysis tubing (Pierce, Rockford Ill.) for 30 hoursagainst three 1 liter changes of phosphate-buffered saline to removeammonium sulfate. The first two liter changes were preserved with 0.02%sodium azide. The final 1 liter buffer change contained no preservative.The dialysate was collected and centrifuged again to remove anyremaining debris at 3000×g for 30 minutes. The antibody solution wasstored at 4° C. for less then 48 hours prior to use. Each sample wasplated on blood agar to verily sterility prior to infusion.

Example 9 Passive Immunization and Challenge

In order to evaluate the protective effect of infused antibody raisedagainst S. aureus proteins expressed during iron-limitation, two groupsof 15 mice each were infused intraperitoneally with either the purifiedantibody preparation (Group 1) or physiological saline (Group 2) in a200 μL infusion. An additional two groups of 15 mice each were infusedsubcutaneously with either the purified antibody preparation (Group 3)or physiological saline (Group 4). After 60 minutes, the 2 groups of 15mice receiving an intraperitoneal infusion were challengedintraperitoneally with 13×10⁸ cfu of S. aureus strain 19636. Similarly,the 2 groups of 15 mice receiving a subcutaneous infusion werechallenged subcutaneously with, 1.3×10⁸ cfu of S. aureus strain 1477 totest for cross-protection against challenge by a different S. aureusstrain. Mortality and/or lesion size was recorded for 5 days and thelivers of all mice were removed post-mortem, homogenized and plated todetermine the number of S. aureus present as a measure of systemicinfection. The Kaplan-Meier survival curves (FIGS. 5 and 6) show theprotective effect afforded by the infusion of antibodies from micevaccinated with the S. aureus proteins expressed during ironrestriction. Although the difference between the infused and controlgroups for the ATCC 19636-challenge groups was not significant (p=0.076,log-rank test), the liver of the single mouse that died within theantibody-infused group at Day 1 was cultured on blood agar to determinethe absence and/or presence of the challenge organism (S. aureus). Theculture derived from this mouse was negative for Staphylococcus andshowed no growth on the blood agar plate or culture medium. In contrast,the livers of the mice that died within the placebo group, were allpositive for the presence of Staphylococcus, in fact, pure cultures wereobtained on each blood agar plate derived from the livers of these mice.While the liver data do not preclude the possibility that the mouse thatdied within the antiody-infused group died of S. aureus infection, theinfection was not systemic, as it was in the placebo group, and themouse may have died for other reasons. Censoring of thisantibody-infused mouse death results in a significant difference betweenantibody-infused and placebo treatments (p=0.015, log-rank test). Thedata for the cross-challenge, where mice were infused with antibodygenerated after vaccination with ATCC 19636-derived proteins andchallenged by S. aureus strain 1477, also showed a protective trend.Between 7 and 14 days post challenge, all mice in the infused andnon-infused groups began to develop necrotic skin lesions. However,gross examination of mice clearly revealed a visible delay in theformation of an observable lesion as well as the severity of the lesionbetween the groups. Infused mice developed lesions more slowly ascompared to non-infused control mice which developed lesion faster theninfused mice and at a greater degree of severity. The infused micehealed faster then non-infused mice. This was clearly evident between 21and 35 days post challenge. Gross examination of mice at 35 days postchallenge showed that non-infused mice were severely disfigured andrevealed a greater degree of scarring. In fact, many of these mice lostnormal posture, in that they appeared twisted in appearance, in contrastto infused mice that did not develop nearly the extensive scar tissueand/or disfigurement as illustrated by the twisted appearance that thenon-infused mice developed. Overall, these data suggest thatinterperitoneal infusion of antibodies raised against S. aureus inducedproteins can both protect against and limit the severity of S. aureusinfection.

Example 10 Evaluation of a Vaccine Composition Derived fromStaphylococcus aureus in a Chronically Infected Dairy Herd

A commercial Dairy herd having a history of chronically high somaticcell counts attributable to Staphylococcus aureus was chosen for theevaluation of a vaccine composition as described in Example 1. Thecriterion for establishing vaccine efficacy of this experimental studywas: 1) decreased incidence of clinical mastitis caused byStaphylococcus aureus among: vaccinates compared to nom-vaccinatedcontrols, 2) improvement (i.e., a decrease) in somatic cell count amongvaccinates compared to controls and 3) decrease in culture positiveisolation rates of S. aureus between vaccinated and non-vaccinatedcontrols. Blood will be taken at the time of the first vaccination (day0) and again at 3 and 6 weeks post initial immunisation. Injection sitereactions or systemic reactions following vaccinations were monitoredthroughout the study. In addition, bulk tank milk samples were culturedand quantitatively enumerated to determine if them was a decrease in thenumber of CFU of Staphylococcus aureus cultured after vaccination.

Three of the Staphylococcus isolates derived from the chronicallyinfected lactating cows within the herd were grown under conditions ofiron-restriction and non-iron restricted conditions as described inExample 1. The three isolates were designated TTX101, TTX102, andTTX103. Extracted samples were examined by SDS-PAGE to compare, bandingprofiles between isolates. Identical banding profiles were observedamong isolates examined; the compositions made from each isolateincluded proteins having molecular weights of 87.73 kDa, 80.46 kDa,65.08 kDa, 54.53 kDa, 37.37 kDa, 35.70 kDa, 34.91 KDa, 33.0 kDa and31.83 kDa. These proteins are the same molecular weights as previouslydescribed in Table 10. In addition, when comparing the isolatesidentical banding profiles were seen with those proteins that wereexpressed in all conditions that were not regulated by iron: 150 kDa,132 kDa, 120 kDa, 75 kDa, 58 kDa, 50 kDa, 44 kDa, 43 kDa, 41 kDa, and 40kDa. These results were consistent with previous observations. Oneisolate designated as TTX101 was chosen as the isolate to manufacture acomposition to be used in this study.

Example 11 Vaccine Preparation of Staphylococcus aureus (TTX101)

A composition was prepared as described in Example 1 using the isolateTTX101. The composition included proteins expressed under iron depleteconditions having molecular weights of 87.73 kDa, 80.46 kDa, 65.08 kDa,54.53 kDa, 37.37 kDa, 35.70 kDa, 34.91 kDa, 33.0 kDa, and 31.83 kDa aswell as non-metal regulated proteins having molecular weights of 150kDa, 132 kDa, 120 kDa, 75 kDa, 58 kDa, 50 kDa, 44 kDa 43 kDa 41 kDa, and40 kDa. The immunizing composition derived from strain TTX101 was usedto prepare the experimental vaccine by emulsifying the extracted proteinsuspension (400 μg total protein per milliliter) into a commercialadjuvant (EMULSIGEN, MVP Laboratories, Ralston Nebr.) using an IKA UltraTurrax T-50 homogenizing vessel (IKA, Cincinnati, Ohio) to give a finaldose of 800 μg total protein in a 2.0 ml injectable volume with anadjuvant concentration of 22.5% vol/vol. The vaccine was administeredsubcutaneously 2 times at 21 day intervals.

Example 12 Experimental Design and Herd Vaccination

Eighteen days before the first vaccination all lactating cows enrolledin the study (N=80) were tested for S. aureus by standardized aerobicbacteriological culture methods by culturing individual milk samplesderived from each lactating cow. In addition, the Somatic Cell Counts(SCC) were enumerated by the Dairy Herd Improvement Association usingstandard methods. Fourteen of the 80 cows were clinically diagnosed withmastitis and were culture positive for S. aureus. The remaining cows(N=66) tested negative for S. aureus. The eighty cows were equallydivided into two groups designated as group-1, vaccinated (N=40) andgroup-2, non-vaccinated (N=40). The fourteen clinically diagnosedStaphylococcus positive cows were equally distributed between bothgroups so that each study group contained 7 cows with clinical mastitis.The average SCC between groups prior to the first vaccination was203,219 in the non-vaccinated controls compared to 240,443 in vaccinates(not statistically different p=0.7).

Eighteen days after the first sampling all cows in group 1 werevaccinated subcutaneously in the upper right shoulder with 2 ml ofvaccine as described in Example 11. Ten days after the first vaccinationmilk samples were taken at this time period by the DHIA for theenumeration of somatic cells from each individual cow. Milk samples werenot bacterioiogically tested at this time period for determining thepresence of Staphylococcus. The difference in the SCC between groups atthis time period was 125.741 (vaccinates) compared to 196,297(controls). This was a 36% difference in the number of somatic cellsbetween vaccinates as compared to non-vaccinated controls. Thedifference in the SCC between the controls and vaccinates at thissampling period was not statistically different (p=0.5). The lack ofstatistical difference in the SCC between groups at both samplingperiods was due to the large variation in individual SCC between cows.The injection site of each vaccinated cow was also examined at this sametime period. None of the cows examined showed any adverse tissuereaction at the site of injection by physical examination. In addition,there was no measurable loss in milk production due to vaccination.

Twenty one days after the first vaccination all cows in group-1(vaccinates) were given their second vaccination or booster. During thetime period between first and second vaccination, cows in both groups(vaccinates and controls) developed tear damage due to a dramatic dropin the environmental temperature resulting in the formation of lesionsat the end of the tear, resulting in the development of infected tearsand potentially increasing the isolation of Staphylococcus duringsampling, which was observed at the third sampling period. Twenty threedays after the second vaccination milk samples were taken by the DHIAfor the enumeration of Somatic Cells from each individual cow. Milksamples were also bacterioiogically tested for the presence ofStaphylococcus aureus. There was a dramatic increase in isolation rareof S. aureus at this time period in the cows that tested negative at thefirst sampling period. In the non-vaccinated controls 42.9% of thesecows now tested positive for S. aureus, in contest to the vaccinates,which only showed and increase of 35.5%. This was a 7.4% differencebetween vaccinates as compared to the non vaccinated controls. It'sdifficult to say that the improvement in the isolation rate of S. aureusin the vaccinated group was due to the effect of the vaccine alone. Onecannot overlook the difficulty in obtaining clean milk samples from cowsthat had tear damage which could increase the potential contamination ofthe milk by S. aureus when obtaining the sample. Nevertheless, there wasa significant difference in the average SCC between vaccinates comparedto controls. The average SCC of the vaccinated group was 222,679compared to 404,278 somatic cells as measured in the control group. Thiswas a 44.9% difference between vaccinates when compared to the nonvaccinated controls. It's interesting to speculate that the differenceseen in the SCC between these groups also coincides with the differencein the isolation rate of S. aureus between groups. However, due to thelarge variation in SCC between individual animals and the small samplesize of the experimental trial in the number of animals the differencewas not statistically different (p=0.28).

At this same time period the injection site of each vaccinated cow wasexamined for any adverse tissue reaction that may have been caused bythe vaccine composition. None of the cows examined showed any adversereaction at the site of injection by physical examination. The vaccinecompositions appeared to be highly tissue compatible and caused nomeasurable loss in milk production after each vaccination.

Monitoring of the cows is continued by measuring SCC and milk samplesfor the presence or absence of Staphylococcus aureus. Some of the cowsof each group are vaccinated a third time at 42 days after the secondvaccination. There appears to be a difference favoring the use of thevaccine composition for decreasing somatic cell counts and controllinginfection caused by Staphylococcus aureus. Further monitoring includesserology based on antibody titers to the vaccine composition, changes inmilk production in vaccinated cows due the improvement in health, anddecreased SCC of vaccinated animals compared to non-vaccinated cohorts.In addition, other experiments are conducted to investigate theprotective index of the vaccine based on dose response followingchallenge with a virulent S. aureus.

Example 13

Since the molecular weights of the proteins among the different S.aureus strains have been demonstrated to be similar and sinceheterologous protection was observed in the mouse challenge study, wesought to determine if the proteins sinning similar molecular weights inFIG. 1 were similar proteins. The technique chosen to characterize theproteins was matrix-assisted laser desorption/ionization massspectrometry (MALDI-MS). A portion of the composition was resolved usingSDS-PAGE as described in Example 1, and the gel was stained withCoomassie Brilliant blue to visualize the proteins.

Materials and Methods

Excision and Washing.

The gel was washed for 10 minutes with water twice. Each protein band ofinterest was excised by cutting as close to the protein band as possibleto reduce the amount of gel present in the sample.

Each gel slice was cut into 1×1 mm cubes and placed in 1.5 ml tube. Thegel pieces were washed with water for 15 minutes. All the solventvolumes used in the wash steps were approximately equal to twice thevolume of the gel slice. The gel slice was next washed withwater/acetonitrile (1:1) for 15 minutes. When the proteins had beenstained with silver, the water/acetonitrile mixture was removed, the gelpieces dried in a SPEEDVAC (ThermoSavant, Holbrook, N.Y.) and thenreduced and alkylated as described below. When the gel pieces were notsilver-stained, the water/acetonitrile mixture was removed, andacetonitrile was added to cover until the gel pieces turned a stickywhite, at which time the acetonitrile was removed. The gel pieces wererehydrated in 100 mM NH₄HCO₃, and after 5 minutes, a volume ofacetonitrile equal to twice the volume of the gel pieces was added. Thiswas incubated for 15 minutes, the liquid removed, and the gel piecesdried in a SPEEDVAC.

Reduction & Alkylation.

The dried gel pieces were rehydrated in 10 mM DTT and 100 mM NH₄HCO₃,and incubated for 45 minutes at 56° C. After allowing the tubes to coolto room temperature, the liquid was removed and the same volume of amixture of 55 mM iodoacetamide and 100 mM NH₄HCO₃ was immediately added.This was incubated for 30 minutes at room temperature in the dark. Theliquid was removed, acetonitrile was added to cover until the gel piecesturned a sticky white, at which time the acetonitrile was removed. Thegel pieces were rehydrated in 100 mM NH₄HCO₃, and after 5 minutes, avolume of acetonitrile equal to twice the volume of the gel pieces wasadded. This was incubated for 15 minutes, the liquid removed, and theget pieces dried in a Speed vac. If the gel was stained with coomasieblue, and residual coomassie still remained, the wash with 100 mMNH₄HCO₃/acetonitrile was repeated.

In-Gel Digestion.

Gel pieces were completely dried down in a Speed Vac. The pieces wererehydrated in digestion buffer (50 mM NH₄HCO₃, 5 mM CaCl₂, 12.5nanograms per microliter (ng/μl) trypsin) at 4° C. Enough buffer wasadded to cover the gel pieces, and more was added as needed. The gelpieces were incubated on ice for 45 minutes, and the supernatant removedand replaced with 5-2 μl of same buffer without trypsin. This wasincubated at 37° C. overnight in an air incubator.

Extraction of Peptides.

A sufficient volume of 25 mM NH₄HCO₃ was added to cover gel pieces, andincubated for 15 minutes (typically in a bath sonicator). The samevolume of acetonitrile was added and incubated for 15 minutes (in a bathsonicator if possible), and the supernatant was recovered. Theextraction was repeated twice, using 5% formic acid instead of NH₄HCO₃.A sufficient volume of 5% formic acid was added to cover gel pieces, andincubated for 15 minutes (typically in a bath sonicator). The samevolume of acetonitrile was added and incubated for 15 minutes (typicallyin a bath sonicator), and the supernatant was recovered. The extractswere pooled, and 10 mM DTT was added to a final concentration of 1 mMDTT. The sample was dried in a SPEEDVAC to a final volume ofapproximately 5 μl.

Desalting of Peptides.

The samples were desalted using a ZIPTIP pipette tips (C18, Millipore,Billerica, Mass.) as suggested by the manufacturer. Briefly, a samplewas reconstituted in reconstitution solution (5:95 acetonitrile:H₂O,0.1%-0.5% trifluoroacetic acid), centrifuged, and the pH checked toverify that it was less than 3. A ZIPTIP was hydrated by aspirating 10μl of solution 1 (50:50 acetonitrile:H₂O, 0.1% trifluorooacetic acid)and discarding the aspirated ailquots. This was followed by aspirating10 μl of solution 2 (0.1% trifluoroacetic acid in deionized H₂O) anddiscarding the aspirated aliquots. The sample was loaded into the tip byaspirating 10 μl of the sample slowly into the tip, expelling it intothe sample tube, and repeating this 5 to 6 times. Ten microliters ofsolution 2 was aspirated into the tip, the solution discarded byexpelling, and this process was repeated 5-7 times to wash. The peptideswere elated by aspirating 2.5 μl of ice cold solution 3 (60:40,acetonitrile:H₂O, 0.1% trifluoroacetic acid), expelling, and thenre-aspirating the same aliquot in and out of the tip 3 times. After thesolution has been expelled from the tip, the tube is capped and storedon ice.

Mass Spectrometric Peptide Mapping.

The peptides were suspended in 10 μl to 30 μl of 5% formic acid, andanalyzed by MALDI-TOF MS (Bruker Daltonics Inc., Billerica, Mass.). Themass spectrum of the peptide fragments was determined as suggested bythe manufacturer. Briefly, a sample containing the peptides resultingfrom a tryptic digest were mixed wish matrix cyano-4-hydroxycinnamicacid, transferred to a target, and allowed to dry. The dried sample wasplaced in the mass spectrometer, irradiated, and the time of flight ofeach ion detected and used to determine a peptide mass fingerprint foreach protein present in the composition. Known polypeptides were used tostandardize the machine.

Data Analysis.

The experimentally observed masses for the peptides in each massspectrum were compared to the expected masses of proteins using thePeptide Mass Fingerprint search method of the Mascot search engine(Matrix Science Ltd., London, UK, see Perkins et al., Electrophoresis20, 3551-3567 (1999)). The search parameters included: database, MSDB orNCBInr; taxonomy, bacteria (eubacteria) or Firmicutes (gram-positivebacteria); type of search, peptide mass fingerprint; enzyme, trypsin;fixed modifications, carbamidomethyl (C) or none; variablemodifications, oxidation (M), carbamidomethyl (C), the combination, ornone; mass values, monoisotopic; protein mass, unrestricted; peptidemass tolerance, between ±150 ppm and ±430 ppm, or ±1 Da; peptide chargestate, Mr; max missed cleavages, 0 or 1; number of queries, 20.

Results

The result of this search was a mass fingerprint for each proteinpresent in the composition is shown in Tables 2, 3, 4, and 5.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forinstance, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProl, PIR, PRF, PDB,and translations from annotated coding regions in Gen-Bank and RefSeq)cited herein are incorporated by reference. The foregoing detaileddescription and examples have been given for clarity of understandingonly. No unnecessary limitations are to be understood therefrom. Theinvention is not limited to the exact details shown and described, forvariations obvious to one skilled to the art will be included within theinvention defined by the claims.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless otherwise indicated to thecontrary, the numerical parameters set forth is the specification andclaims are approximations that may vary depending open the desiredproperties sought to be obtained by the present invention. At the veryleast, and not as an attempt to limit the doctrine of equivalents to thescope of the claims, each numerical parameter should at least beconstrued in light of the number of reported significant digits and byapplying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific crumples am reported as precisely aspossible. All numerical values, however, inherently contain a rangenecessarily resulting from the standard deviation found in theirrespective testing measurements.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

What is claimed is:
 1. A method comprising: administering an effectiveamount of a composition to a subject at risk of developing lesionscaused by infection by Staphylococcus aureus, the compositioncomprising: at least five isolated polypeptides having molecular weightsof 88 kDa, 55 kDa, 38 kDa, 37 kDa, 36 kDa, 35 kDa, or 33 kDa, whereinmolecular weight is determined by electrophoresis on a sodium dodecylsulfate-polyacrylamide gel; wherein the isolated polypeptide having amolecular weight of 88 kDa comprises an amino acid sequence having atleast 95% identity to the amino acid sequence of a referencepolypeptide, wherein the reference polypeptide has a molecular weight of88 kDa as determined by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis, comprises the amino acid sequences depicted in SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33, and is expressed byStaphylococcus aureus ATCC strain 19636 at a greater level when grown inmedium comprising 1600 μM 2,2-dipyridyl compared to when grown in themedium without the 2,2-dipyridyl; wherein the isolated polypeptidehaving a molecular weight of 55 kDa comprises an amino acid sequencehaving at least 95% identity to the amino acid sequence of a referencepolypeptide, wherein the reference polypeptide has a molecular weight of55 kDa as determined by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis, comprises the amino acid sequences depicted in SEQ IDNO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ IDNO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ IDNO:49, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ IDNO:57, SEQ ID NO:58, and SEQ ID NO:59, and is expressed byStaphylococcus aureus ATCC strain 19636 at a greater level when grown inmedium comprising 1600 μM 2,2-dipyridyl compared to when grown in themedium without the 2,2-dipyridyl; wherein the isolated polypeptidehaving a molecular weight of 38 kDa comprises an amino acid sequencehaving at least 95% identity to the amino acid sequence of a referencepolypeptide, wherein the reference polypeptide has a molecular weight of38 kDa as determined by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis, comprises the amino acid sequences depicted in SEQ IDNO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:69, SEQ IDNO:70, SEQ ID NO:72, SEQ ID NO:75, SEQ ID NO:76, and SEQ ID NO:77, andis expressed by Staphylococcus aureus ATCC strain 19636 at a greaterlevel when grown in medium comprising 1600 μM 2,2-dipyridyl compared towhen grown in the medium without the 2,2-dipyridyl; wherein the isolatedpolypeptide having a molecular weight of 37 kDa comprises an amino acidsequence having at least 95% identity to the amino acid sequence of areference polypeptide, wherein the reference polypeptide has a molecularweight of 37 kDa as determined by sodium dodecyl sulfate-polyacrylamidegel electrophoresis, comprises the amino acid sequences depicted in SEQID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ IDNO:87, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:94, SEQ IDNO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:100, and SEQID NO:101, and is expressed by Staphylococcus aureus ATCC strain 19636at a greater level when grown in medium comprising 1600 μM 2,2-dipyridylcompared to when grown in the medium without the 2,2-dipyridyl; whereinthe isolated polypeptide having a molecular weight of 36 kDa comprisesan amino acid sequence having at least 95% identity to the amino acidsequence of a reference polypeptide, wherein the reference polypeptidehas a molecular weight of 36 kDa as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis, comprises the amino acidsequences depicted in SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, and SEQ IDNO:110, and is expressed by Staphylococcus aureus ATCC strain 19636 at agreater level when grown in medium comprising 1600 μM 2,2-dipyridylcompared to when grown in the medium without the 2,2-dipyridyl; whereinthe isolated polypeptide having a molecular weight of 35 kDa comprisesan amino acid sequence having at least 95% identity to the amino acidsequence of a reference polypeptide, wherein the reference polypeptidehas a molecular weight of 35 kDa as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis, comprises the amino acidsequences depicted in SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119,SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, and SEQ IDNO:124, and is expressed by Staphylococcus aureus ATCC strain 19636 at agreater level when grown in medium comprising 1600 μM 2,2-dipyridylcompared to when grown in the medium without the 2,2-dipyridyl; andwherein the isolated polypeptide having a molecular weight of 33 kDacomprises an amino acid sequence having at least 95% identity to theamino acid sequence of a reference polypeptide, wherein the referencepolypeptide has a molecular weight of 33 kDa as determined by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis, comprises the aminoacid sequences depicted in SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128,SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ IDNO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQID NO:140, SEQ ID NO:142, and SEQ ID NO:143, and is expressed byStaphylococcus aureus ATCC strain 19636 at a greater level when grown inmedium comprising 1600 μM 2,2-dipyridyl compared to when grown in themedium without the 2,2-dipyridyl.
 2. The method of claim 1 wherein thesubject is a mammal.
 3. The method of claim 2 wherein the mammal is ahuman.
 4. The method of claim 2 wherein the mammal is bovine, ovine,porcine, caprine, cervine, bisontine, or equine.
 5. The method of claim1 wherein the subject is avian.
 6. The method of claim 1 wherein thesubject is a companion animal.
 7. The method of claim 1 wherein thecomposition is administered prophylactically.
 8. The method of claim 1wherein an effective amount is an amount effective to decrease theaverage size of lesions exhibited by the subject compared to anuntreated control.
 9. The method of claim 1 wherein an effective amountis an amount effective to decrease the number of lesions exhibited bythe subject compared to an untreated control.
 10. The method of claim 1wherein the lesion comprises a necrotic skin lesion.
 11. The method ofclaim 1 wherein the composition comprises the 88 kDa polypeptide, the 55kDa polypeptide, the 38 kDa polypeptide, the 37 kDa polypeptide, the 36kDa polypeptide, the 35 kDa polypeptide, and the 33 kDa polypeptide. 12.A method comprising: administering an effective amount of a compositionto a subject at risk of developing a post-surgical wound infectioncaused by Staphylococcus aureus, the composition comprising: at leastfive isolated polypeptides having molecular weights of 88 kDa, 55 kDa,38 kDa, 37 kDa, 36 kDa, 35 kDa, or 33 kDa, wherein molecular weight isdetermined by electrophoresis on a sodium dodecyl sulfate-polyacrylamidegel; wherein the isolated polypeptide having a molecular weight of 88kDa comprises an amino acid sequence having at least 95% identity to theamino acid sequence of a reference polypeptide, wherein the referencepolypeptide has a molecular weight of 88 kDa as determined by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis, comprises the aminoacid sequences depicted in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21,SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27,SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32,and SEQ ID NO:33, and is expressed by Staphylococcus aureus ATCC strain19636 at a greater level when grown in medium comprising 1600 μM2,2-dipyridyl compared to when grown in the medium without the2,2-dipyridyl; wherein the isolated polypeptide having a molecularweight of 55 kDa comprises an amino acid sequence having at least 95%identity to the amino acid sequence of a reference polypeptide, whereinthe reference polypeptide has a molecular weight of 55 kDa as determinedby sodium dodecyl sulfate-polyacrylamide gel electrophoresis, comprisesthe amino acid sequences depicted in SEQ ID NO:35, SEQ ID NO:37, SEQ IDNO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ IDNO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:51, SEQ IDNO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:58, and SEQID NO:59, and is expressed by Staphylococcus aureus ATCC strain 19636 ata greater level when grown in medium comprising 1600 μM 2,2-dipyridylcompared to when grown in the medium without the 2,2-dipyridyl; whereinthe isolated polypeptide having a molecular weight of 38 kDa comprisesan amino acid sequence having at least 95% identity to the amino acidsequence of a reference polypeptide, wherein the reference polypeptidehas a molecular weight of 38 kDa as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis, comprises the amino acidsequences depicted in SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ IDNO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:75, SEQ IDNO:76, and SEQ ID NO:77, and is expressed by Staphylococcus aureus ATCCstrain 19636 at a greater level when grown in medium comprising 1600 μM2,2-dipyridyl compared to when grown in the medium without the2,2-dipyridyl; wherein the isolated polypeptide having a molecularweight of 37 kDa comprises an amino acid sequence having at least 95%identity to the amino acid sequence of a reference polypeptide, whereinthe reference polypeptide has a molecular weight of 37 kDa as determinedby sodium dodecyl sulfate-polyacrylamide gel electrophoresis, comprisesthe amino acid sequences depicted in SEQ ID NO:80, SEQ ID NO:82, SEQ IDNO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:89, SEQ IDNO:90, SEQ ID NO:91, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ IDNO:97, SEQ ID NO:99, SEQ ID NO:100, and SEQ ID NO:101, and is expressedby Staphylococcus aureus ATCC strain 19636 at a greater level when grownin medium comprising 1600 μM 2,2-dipyridyl compared to when grown in themedium without the 2,2-dipyridyl; wherein the isolated polypeptidehaving a molecular weight of 36 kDa comprises an amino acid sequencehaving at least 95% identity to the amino acid sequence of a referencepolypeptide, wherein the reference polypeptide has a molecular weight of36 kDa as determined by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis, comprises the amino acid sequences depicted in SEQ IDNO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQID NO:108, SEQ ID NO:109, and SEQ ID NO:110, and is expressed byStaphylococcus aureus ATCC strain 19636 at a greater level when grown inmedium comprising 1600 μM 2,2-dipyridyl compared to when grown in themedium without the 2,2-dipyridyl; wherein the isolated polypeptidehaving a molecular weight of 35 kDa comprises an amino acid sequencehaving at least 95% identity to the amino acid sequence of a referencepolypeptide, wherein the reference polypeptide has a molecular weight of35 kDa as determined by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis, comprises the amino acid sequences depicted in SEQ IDNO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121,SEQ ID NO:122, SEQ ID NO:123, and SEQ ID NO:124, and is expressed byStaphylococcus aureus ATCC strain 19636 at a greater level when grown inmedium comprising 1600 μM 2,2-dipyridyl compared to when grown in themedium without the 2,2-dipyridyl; and wherein the isolated polypeptidehaving a molecular weight of 33 kDa comprises an amino acid sequencehaving at least 95% identity to the amino acid sequence of a referencepolypeptide, wherein the reference polypeptide has a molecular weight of33 kDa as determined by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis, comprises the amino acid sequences depicted in SEQ IDNO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQID NO:131, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136,SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, and SEQ IDNO:143, and is expressed by Staphylococcus aureus ATCC strain 19636 at agreater level when grown in medium comprising 1600 μM 2,2-dipyridylcompared to when grown in the medium without the 2,2-dipyridyl.
 13. Themethod of claim 12 wherein the subject is a mammal.
 14. The method ofclaim 13 wherein the mammal is a human.
 15. The method of claim 13wherein the mammal is bovine, ovine, porcine, caprine, cervine,bisontine, or equine.
 16. The method of claim 12 wherein the subject isavian.
 17. The method of claim 12 wherein the subject is a companionanimal.
 18. The method of claim 12 wherein the composition isadministered prophylactically.
 19. The method of claim 12 wherein thecomposition comprises the 88 kDa polypeptide, the 55 kDa polypeptide,the 38 kDa polypeptide, the 37 kDa polypeptide, the 36 kDa polypeptide,the 35 kDa polypeptide, and the 33 kDa polypeptide.